CN108026153A - The alternatively new exendin-4 derivative of the dual GLP-1/ glucagon receptors activator of property peptide - Google Patents

Abstract

The present invention relates to dual 1/ glucagon receptor activators of GLP and its for example in treatment Metabolic Syndrome illness, including diabetes and it is fat in, and for reducing the medical usage of excessive food intake.

Description

New monster exosomes as selective peptide dual GLP-1/glucagon receptor agonists secretin-4 derivatives

describe

field of invention

The present invention relates to dual GLP-1/glucagon receptor agonists and their medical use, for example, in the treatment of metabolic syndrome disorders, including diabetes and obesity, and for reducing excess food intake. These dual GLP-1/glucagon receptor agonists show reduced activity at the GIP receptor to reduce the risk of hypoglycemia. They are structurally derived from exendin-4 and display high solubility and stability under acidic conditions in the presence of antimicrobial preservatives such as m-cresol or phenol, making them particularly suitable for use with other Combination of antidiabetic compounds.

Background of the invention

Glucagon-like peptide-1 (GLP -1) and dual agonists of the glucagon receptor, for example by combining the actions of GLP-1 and glucagon in one molecule, which leads to an antidiabetic and antidiabetic effect superior to pure GLP-1 agonists Therapeutic principles for pronounced weight-lowering effects, especially due to increased satiety and energy expenditure mediated by the glucagon receptor.

Holst (Physiol.Rev.2007,87,14091439) and Meier (Nat.Rev.Endocrinol.2012,8,728-742) describe GLP-1 receptor agonists, such as GLP-1, liraglutide and Exendin-4 (Exendin-4) has 3 major pharmacological activities to improve glycemic control in T2DM (type 2 diabetes) patients by reducing fasting and postprandial glucose (FPG and PPG): (i) increasing Glucose-dependent insulin secretion (improved first and second phases), (ii) glucagon-inhibitory activity under hyperglycemic control, and (iii) delayed gastric emptying rate, resulting in a delay of meal-derived glucose absorb.

The amino acid sequence of GLP-1(7-36)-amide is shown in SEQ ID NO:1.

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH ₂

Lixisenatide is a marketed chemically modified GLP-1 analog in which, among other modifications, a fatty acid is linked to a lysine in position 20, resulting in a prolonged duration of action (Drucker et al., Nat. Rev. Drug Disc. 2010, 9, 267-268; Buse et al., Lancet 2009, 374, 39-47).

The amino acid sequence of lixisenatide is shown in SEQ ID NO:2.

HAEGTFTSDVSSYLEGQAAK((S)-4-Carboxy-4-hexadecanoylamino-butyryl-)EFIAWLVRGRG-OH

Glucagon is a 29 amino acid peptide that is released into the bloodstream when circulating glucose is low. The amino acid sequence of glucagon is shown in SEQ ID NO:3.

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH

During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing blood glucose levels to rise to normal levels. Recent publications suggest that glucagon additionally has beneficial effects on reduction of body fat mass, reduction of food intake, and increase of energy expenditure (see eg Heppner et al., Physiology & Behavior 2010, 100, 545-548).

GIP (glucose-dependent insulinotropic polypeptide) is a 42 amino acid peptide that is released from intestinal K cells after food intake. GIP and GLP-1 are two enteroendocrine cell-derived hormones of the gut that are responsible for the incretin effects that account for more than 70% of the insulin response to an oral glucose challenge (Baggio et al. Gastroenterology 2007, 132 , 2131–2157).

The amino acid sequence of GIP is shown in SEQ ID NO:5.

YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH

Based on the structure of GLP-1 or glucagon, and binds and activates both glucagon and GLP-1 receptors (Hjorth et al. Journal of Biological Chemistry, 1994, 269, 30121-30124; Day et al., Nature Chem Biol, 2009,5,749-757) and peptides that inhibit weight gain and reduce food intake are described in patent applications WO2008/071972, WO 2008/101017, WO 2009/155258, WO 2010/096052, WO 2010/096142, WO2011/075393, WO 2008 /152403, WO 2010/070251, WO 2010/070252, WO 2010/070253, WO2010/070255, WO 2011/160630, WO 2011/006497, WO 2011/087671, WO 2011/087672, WO20411/16 Wo 2012/150503, wo2012/158965, wo 2012/1774444, wo2013/004983, wo 2013/092703, wo2014/017843, wo2014/041195, wo 2014/041375, wo2014/081402222, wo2014/14/1702222, wo2014/1402222, wo2014/1402222, wo2014/1402222, wo2014/1402222, wo2014/1402222, wo2014/1402222 055801, WO2015/055802, WO 2015/132599, WO 2016/045400, WO 2016/055610 and WO 2016/065090, the contents of which are incorporated herein by reference. Other peptide dual agonists are described in WO 2012/177443, WO2013/186240, WO2014/056872, WO2014/091316, WO 2015/086731, WO 2015/086732 and WO2015/086733, the contents of which are incorporated herein by reference. Body weight reduction was shown to be superior to pure GLP-1 agonists.

Exendin-4 is a 39 amino acid peptide produced by the salivary glands of the Gila monster (Heloderma suspectum) (Eng et al., J. Biol. Chem. 1992, 267 ,7402-7405). Exendin-4 is an activator of the GLP-1 receptor, whereas it showed low activation of the GIP receptor and did not activate the glucagon receptor (see Table 1).

Table 1 : Potency of exendin-4 at increasing concentrations on human GLP-1, GIP and glucagon receptors (indicated in pM) and measured cAMP formed as described in Methods.

The amino acid sequence of exendin-4 is shown in SEQ ID NO:4.

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

Exendin-4 shares many of the observed glucose-regulating effects with GLP-1. Clinical and nonclinical studies have shown that exendin-4 has several beneficial antidiabetic properties, including glucose-dependent enhancement of insulin synthesis and secretion, glucose-dependent inhibition of glucagon secretion, slowing of gastric Emptying, decreased food intake and body weight, and increases in markers of beta-cell mass and beta-cell function (Gentilella et al., Diabetes Obes Metab., 2009, 11, 544-556; Norris et al., Diabet Med., 2009, 26, 837-846; Bunck et al., Diabetes Care., 2011, 34, 2041-2047).

These effects are beneficial not only for diabetics, but also for patients suffering from obesity. Patients with obesity have a higher risk of diabetes, hypertension, hyperlipidemia, cardiovascular and musculoskeletal diseases.

Exendin-4 is resistant to cleavage by dipeptidyl peptidase-4 (DPP4) relative to GLP-1, resulting in a longer half-life and duration of action in vivo (Eng J., Diabetes, 1996, 45 (Suppl 2):152A (Abstract 554)).

It has also been shown that exendin-4 is much more stable against neutral endopeptidase (NEP) degradation when compared to GLP-1, glucagon, or oxyntomodulin (Druce et al., Endocrinology, 2009, 150(4), 1712-1722). However, exendin-4 is chemically unstable due to the oxidation of methionine at position 14 (Hargrove et al., Regul. Pept., 2007, 141, 113-119) and the desaturation of asparagine at Amides and isomerization (WO 2004/035623).

Bloom et al. (WO 2006/134340) disclosed that peptides that bind and activate both the glucagon and GLP-1 receptors can be constructed as hybrid molecules from glucagon and exendin-4, where N The terminal portion (eg residues 1-14 or 1-24) is derived from glucagon and the C-terminal portion (eg residues 15-39 or 25-39) is derived from exendin-4. Such peptides comprise the amino acid motif YSKY of glucagon in positions 10-13. Krstenansky et al. (Biochemistry, 1986, 25, 3833-3839) showed the importance of these residues 10-13 of glucagon for its receptor interaction and activation of adenylyl cyclase.

In the exendin-4 derivatives described in the present invention several of the underlying residues differ from glucagon and the peptides described in WO 2006/134340. In particular, residues Tyr10 and Tyr13 (known to contribute to fibrillation of glucagon) (JS Pedersen et al., Biochemistry, 2006, 45, 14503-14512) were replaced by Leu at position 10 and Leu at position 13 Gln in (a non-aromatic polar amino acid). This substitution (especially in combination with isoleucine at position 23 and glutamic acid at position 24) leads to exendin-4 derivatives with potentially improved biophysical properties, such as aggregation behavior or solubility in solution . Non-conservative substitution of an aromatic amino acid with a polar amino acid in position 13 of an exendin-4 analog leads to a peptide with high activity at the glucagon receptor, retaining its activity at the GLP-1 receptor activity (see also WO2013/186240 and WO2014/056872).

Summary of the invention

Native exendin-4 is a pure GLP-1 receptor agonist with no activity at the glucagon receptor and low activity at the GIP receptor. Provided herein are exendin-4 derivatives that are based on the structure of native exendin-4, but differ at more than 14 positions from SEQ ID NO:4, wherein the differences contribute to pancreatic hypertrophy Enhancement of agonist activity on glucagon receptors. Among other substitutions, the methionine at position 14 was replaced with an amino acid carrying a _-NH2 group in the side chain, which was further substituted with a lipophilic residue (such as a fatty acid in combination with a linker). Furthermore, surprisingly, we found that Aib amino acids at positions 27 and 34, Pro at positions 32, and Lys at positions 35 and 39 at 27, Substitution of exendin-4 amino acids at positions 32, 34, 35 and 39 resulted in reduced GIP receptor activity. Reduced activation of the GIP receptor is potentially beneficial as it has been reported in the literature that high levels of GIP in diabetic patients can lead to more frequent hypoglycemic episodes in some cases (McLaughlin et al., J Clin Endocrinol Metab, 2010, 95, 1851 – 1855; Hadji-Georgopoulos, J Clin Endocrinol Metab, 1983, 56, 648-652).

The present invention provides a peptide compound having formula (I) or a salt or solvate thereof:

H ₂ N-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Asp-Glu-Gln-Arg-Ala-Lys-Leu-Phe-Ile- Glu-Trp-Leu-Aib-X28-X29-Gly-Pro-Pro-Ser-Aib-Lys-Pro-Pro-Pro-Lys-R ¹ (I)

in

X14 represents an amino acid residue with a functionalized -NH ₂ side chain group selected from the group consisting of Lys, Orn, Dab or Dap, wherein the -NH ₂ side chain group passes through -ZC(O)-R ⁵ functionalized, where

Z represents the linker in all stereoisomeric forms, and

^R is a module comprising up to 50 carbon atoms and heteroatoms selected from N and O,

X28 represents an amino acid residue selected from Ala, Lys and Ser,

X29 represents an amino acid residue selected from D-Ala and Gly,

^R1 is _NH2 or OH.

The compounds of the invention are GLP-1 and glucagon receptor agonists, as determined by the observation that they are able to stimulate intracellular cAMP formation in the assay system described in the methods.

According to another embodiment, the compounds of the invention (in particular with further substitution of lysine 14 to a lipophilic residue) exhibit at least 0.1% (i.e. _EC50 <700pM), more preferably 1% (i.e. _EC50 <70pM), more preferably 5% (i.e. _EC50 <14pM) and even more preferably 10% (i.e. _EC50 <7pM) relative activity . In addition, the compound exhibits at least 0.1% (i.e. _EC50 <500pM), more preferably 0.25% (i.e. _EC50 <200pM) and even more preferably 0.5% (i.e. EC50<200pM) at the glucagon receptor compared to native glucagon ( ie EC ₅₀ <100 pM) relative activity.

In a specific embodiment, the peptidic compound has a relative activity at the glucagon receptor of at least 0.09% compared to native glucagon.

In a further specific embodiment, the peptidic compound has at least 0.1% relative activity at the GLP-1 receptor compared to GLP1(7-36)-amide.

As used herein, the term "activity" preferably refers to the ability of a compound to activate the human GLP-1 receptor and the human glucagon receptor. More preferably, the term "activity" as used herein refers to the ability of a compound to stimulate intracellular cAMP formation. As used herein, the term "relative activity" is understood to mean the ability of a compound to activate a receptor at a certain ratio compared to another receptor agonist or compared to another receptor. Activation of the receptor by an agonist (for example by measuring cAMP levels) is determined as described herein, for example as described in Example 5.

The compound of the present invention preferably has 450pmol or less, preferably 200pmol or less, more preferably 100pmol or less, more preferably 50pmol or less, more preferably 25pmol or less, more preferably 10pmol or less, more preferably 8pmol or less Small, and more preferably 5 pmol or less EC ₅₀ on hGLP-1 receptors and/or 500 pmol or less, preferably 300 pmol or less, more preferably 200 pmol or less, more preferably 150 pmol or less, more preferably 100 pmol Or less, more preferably 75pmol or less of the EC ₅₀ and/or 750pmol or more of hGlucagon (hGlucagon) receptors, preferably 1500pmol or more; more preferably 2000pmol or more of hGIP receptors The _EC50 . It is particularly preferred that the _EC50 for both hGLP-1 and hglucagon receptors is 250 pmol or less, more preferably 200 pmol or less, more preferably 150 pmol or less, more preferably 100 pmol or less, more preferably 100 pmol or less, more preferably Preferably 75 pmol or less. _EC50 for hGLP-1 receptor, hglucagon receptor and hGIP receptor can be determined as described in the methods herein and as used to generate the results described in Example 5, Table 5.

Peptide compounds of formula (I), especially those having a 14th lysine further substituted as a lipophilic residue, are the same as those having an initial methionine (from exendin-4) or a 14th lysine Derivatives of leucine (see also WO2014/056872) show increased activation of the glucagon receptor in comparison. Furthermore, oxidation of methionine (in vitro or in vivo) is no longer possible. In addition, this modification leads to an improved pharmacokinetic profile.

Surprisingly, it was found that, compared to the corresponding derivatives with amino acids of native exendin-4 in the corresponding positions (Lys27, Ser32, Gly34, Ala35, Ser39), carrying Compounds of formula (I) with Aib amino acid at position 32, Pro at position 32 and Lys at position 35 and 39 showed reduced activity at the GIP receptor, as shown in Example 6, Table 6. Reduced activation of the GIP receptor is potentially beneficial as it has been reported in the literature that high levels of GIP in diabetic patients can in some cases lead to more frequent hypoglycemic episodes (T McLaughlin et al., J Clin Endocrinol Metab, 95, 1851-1855 , 2010; A Hadji-Georgopoulos, J Clin Endocrinol Metab, 56, 648-652, 1983).

In one embodiment, the compounds of the invention have high solubility at acidic and/or physiological pH values at 25°C or 40°C, for example at pH 4 to 5, in the presence of antimicrobial preservatives, such as phenol or m-cresol , especially the acidity range of pH 4.5 and/or the more physiological range of pH 6 to 8, especially at pH 7.4, in another embodiment at least 1 mg/ml and in a particular embodiment at least 5 mg/ml ml.

Furthermore, the compounds of the present invention preferably have high stability when stored in solution in the presence of antimicrobial preservatives, such as phenol or m-cresol. Preferred assay conditions for determining stability are in the acidity range of pH 4 to 5, especially pH 4.5 and/or the more physiological range of pH 6 to 8, especially pH 7.4 in solution at 25° C. or 40 Store at ℃ for 7 days. Peptide stability was determined by chromatographic analysis as described in Methods. Preferably, the loss of purity does not exceed 20%, more preferably does not exceed 15%, even more preferably does not exceed 10%, and even more preferably does not exceed 5% after 7 days at 40°C in a solution at pH 4.5 or pH 7.4.

In one embodiment, the compound of the invention is present at a concentration of ≥ 7 mg/ml in the presence of an antimicrobial preservative, such as phenol or m-cresol, for example in the acidity range of pH 4 to 5, especially pH 4.5 and/or Or the more physiological range of pH 6 to 8, especially pH 7.4 exhibits a hydrodynamic diameter of 10 nm or less at 25°C, as determined by dynamic light scattering, as described in Methods.

Compounds of the invention are more resistant to cleavage by neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP4) when compared to native GLP-1 and glucagon, resulting in longer Half-life and duration of action in vivo.

Preferably, the compounds of the invention comprise a peptide moiety which is a linear sequence of 39 aminocarboxylic acids, in particular α-aminocarboxylic acids linked by peptide, ie carboxamide, bonds.

In one embodiment, ^R1 is _NH2 .

The _-NH2 side chain group at position X14 is functionalized by -ZC(O) ^-R5 , where ^R5 is a module comprising up to 50 carbon atoms and optionally heteroatoms independently selected from N and O.

Specific preferred examples of -ZC(O) ^-R groups are listed in Table 2 below, which are selected from:

(S)-4-carboxy-4-hexadecanoylamino-butyryl-, (S)-4-carboxy-4-octadecanoylamino-butyryl-, (S)-4-carboxy-4-(( S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-(2-{2-[(4S)-4-carboxy-4-deca Hexaacylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl-, (2-{2-[2-(2- {2-[(4S)-4-Carboxyl-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl Base-, [2-(2-{2-[2-(2-{2-[2-(2-octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy )-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-.

Further preference is given to stereoisomers, especially the enantiomers, the S- or R-enantiomers, of these groups. The term "R" in Table 2 is intended to refer to the -ZC(O)-R ⁵ attachment site on the peptide backbone, eg the ε-amino group of Lys.

Table 2

A further embodiment relates to the group of compounds of formula (I), wherein

X14 represents Lys, wherein said _-NH2 side chain group is functionalized with group -ZC(O) ^R5 , wherein

Z represents a group selected from the group consisting of gGlu, gGlu-gGlu, AEEAc-AEEAc-gGlu and AEEAc-AEEAc-AEEAc, and

R ⁵ represents a group selected from pentadecyl or heptadecyl.

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys in which the -NH _side chain group is functionalized by (S)-4-carboxy-4-octadecanoylamino-butyryl,

X28 represents an amino acid residue selected from Ala and Lys,

X29 means Gly,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys in which the _-NH2 side chain group is functionalized by (S)-4-carboxy-4-hexadecanoylamino-butyryl,

X28 means Ala,

X29 means Gly,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys in which the _-NH2 side chain group is functionalized by (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-functionalized,

X28 represents an amino acid residue selected from Ala, Ser and Lys,

X29 represents an amino acid residue selected from Gly and D-Ala,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys, where the _-NH2 side chain group is functionalized by: (S)-4-carboxy-4-hexadecanoylamino-butyryl-, (S)-4-carboxy-4-octadecanoylamino -butyryl-,(S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,(2-{2-[2-( 2-{2-[(4S)-4-Carboxyl-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy) -Acetyl-,(2-{2-[2-(2-{2-[(4S)-4-carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy) -acetylamino]-ethoxy}-ethoxy)-acetyl-,[2-(2-{2-[2-(2-{2-[2-(2-octadecanoylamino-ethyl Oxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,

X28 means Ala,

X29 represents an amino acid residue selected from D-Ala and Gly,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys in which the _-NH2 side chain group is functionalized by (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-functionalized,

X28 means Ser,

X29 represents an amino acid residue selected from D-Ala and Gly,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys, wherein -NH _The side chain group is passed through (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (S )-4-carboxy-4-octadecanoylamino-butyryl-functionalized,

X28 means Lys,

X29 represents an amino acid residue selected from D-Ala and Gly,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys in which the _-NH2 side chain group is functionalized by (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-functionalized,

X28 represents an amino acid residue selected from Ala, Lys and Ser,

X29 means D-Ala,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys, where the -NH _side chain group is functionalized by: (S)-4-carboxy-4-hexadecanoylamino-butyryl-, (S)-4-carboxy-4-octadecanoylamino -butyryl-, (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-( 2-{2-[(4S)-4-Carboxyl-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy) -Acetyl-, (2-{2-[2-(2-{2-[(4S)-4-carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy) -acetylamino]-ethoxy}-ethoxy)-acetyl-, [2-(2-{2-[2-(2-{2-[2-(2-octadecanoylamino-ethyl Oxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,

X28 represents an amino acid residue selected from Ala, Lys and Ser,

X29 means Gly,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys in which the _-NH2 side chain group is functionalized by (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-functionalized,

X28 means Ala,

X29 represents an amino acid residue selected from Gly and D-Ala,

R ¹ represents NH ₂ .

A further embodiment relates to the group of compounds of formula (I) or their salts or solvates, wherein

X14 represents Lys, where the -NH _side chain group is functionalized by: (S)-4-carboxy-4-hexadecanoylamino-butyryl-, (S)-4-carboxy-4-octadecanoylamino -butyryl-, (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-.

Yet another embodiment relates to the group of compounds of formula (I) or salts or solvates thereof, wherein

X14 represents Lys in which the _-NH2 side chain group is functionalized by (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-.

Specific examples of peptide compounds of formula (I) are compounds of SEQ ID NO: 6-17, and salts or solvates thereof.

Specific examples of peptide compounds of formula (I) are SEQ ID NO: 6 and 7, and salts or solvates thereof.

According to a specific embodiment, the compound of the invention is represented by SEQ ID NO.: 6, or a salt or solvate thereof.

According to another specific embodiment, the compound of the invention is represented by SEQ ID NO.: 7, or a salt or solvate thereof.

According to another specific embodiment, the compound of the invention is represented by SEQ ID NO.: 8, or a salt or solvate thereof.

According to another specific embodiment, the compound of the invention is represented by SEQ ID NO.: 9, or a salt or solvate thereof.

According to another specific embodiment, the compound of the invention is represented by SEQ ID NO.: 10, or a salt or solvate thereof.

According to another specific embodiment, the compound of the invention is represented by SEQ ID NO.: 11, or a salt or solvate thereof.

According to another specific embodiment, the compound of the invention is represented by SEQ ID NO.: 15, or a salt or solvate thereof.

In certain embodiments, i.e. when the compound of formula (I) comprises genetically encoded amino acid residues, the invention further provides a nucleic acid (which may be DNA or RNA) encoding said compound, comprising expression of said nucleic acid Vectors, and host cells containing the nucleic acid or expression vectors.

In a further aspect, the invention provides a compound of formula (I) for use in medicine, especially in human medicine. In certain embodiments, compounds of formula (I) are for use as medicaments.

In a further aspect, the invention provides compositions comprising a compound of the invention in admixture with a carrier. In preferred embodiments, the composition is a pharmaceutically acceptable composition, and the carrier is a pharmaceutically acceptable carrier. The compounds of the invention may be in the form of salts, eg pharmaceutically acceptable salts, or solvates, eg hydrates. In yet another aspect, the invention provides compositions for use in a method of medical treatment, especially in human medicine.

In certain embodiments, nucleic acids or expression vectors can be used as therapeutic agents, eg, in gene therapy.

The compounds of formula (I) are suitable for therapeutic use without additional therapeutically effective agents. However, in other embodiments, the compounds may be used together with at least one additional therapeutically active agent, as described under "Combination Therapies".

Compounds of formula (I) are particularly suitable for the treatment or prophylaxis of disorders caused by, associated with and/or accompanied by disturbances in carbohydrate and/or lipid metabolism. Disturbed diseases or conditions, for example for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity and metabolic syndrome. Furthermore, the compounds according to the invention are particularly suitable for the treatment or prophylaxis of degenerative diseases, especially neurodegenerative diseases.

The compounds described find use inter alia in preventing weight gain or promoting weight loss. "Prevention" means inhibition or reduction when compared to the absence of treatment, and does not necessarily imply complete cessation of a condition.

Compounds of the invention may cause a decrease in food intake and/or an increase in energy expenditure, leading to the observed effects on body weight.

Independent of their effect on body weight, the compounds of the invention may have a beneficial effect on circulating cholesterol levels, be able to improve lipid levels, particularly LDL, and HDL levels (eg increase HDL/LDL ratio).

Accordingly, the compounds of the present invention can be used in the direct or indirect treatment of any condition caused by or characterized by excess body weight, such as the treatment and/or prevention of obesity, morbid obesity, obesity-associated inflammation, obesity-associated gallbladder disease, obesity-induced of sleep apnea. They may also be used in the treatment and prevention of metabolic syndrome, diabetes, hypertension, atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease or stroke. Their effect in these conditions may be due to or related to their effect on body weight, or independent of it.

Preferred medicinal uses include delaying or preventing disease progression in type 2 diabetes, treating metabolic syndrome, treating obesity or preventing overweight, for reducing food intake, increasing energy expenditure, reducing body weight, delaying progression from impaired glucose tolerance (IGT) Progression to type 2 diabetes; delays progression from type 2 diabetes to insulin-requiring diabetes; regulates appetite; induces satiety; prevents weight regain following successful weight loss; treats diseases or conditions associated with overweight or obesity; treats appetite Hypertension; treatment of binge eating; treatment of atherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia, coronary heart disease, hepatic steatosis, treatment of beta-blocker poisoning, used to suppress gastric Bowel Motility Uses.

In addition, the compounds can be used in the investigation of the gastrointestinal tract using a combination of techniques such as X-ray, CT and NMR scans.

Further preferred medical uses include the treatment or prevention of degenerative disorders, especially neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, ataxia, Examples include spinocerebellar ataxia, Kennedy disease, myotonic dystrophy, dementia with Lewy bodies, multiple system atrophy, amyotrophic lateral sclerosis, primary lateral sclerosis, spinal Muscular atrophy, prion-associated diseases such as Creutzfeldt-Jacob disease, multiple sclerosis, telangiectasia, Batten disease, corticobasal degeneration, subacute combined spinal cord Degeneration, Tabes dorsalis, Tay-Sachs disease, Toxic encephalopathy, Infantile Refsum disease, Refsum disease, Neuroacanthocytosis, Niemann-Petid Niemann-Pick disease, Lyme disease, Machado-Joseph disease, Sandhoff disease, Shy-Drager syndrome syndrome), wobbly hedgehog syndrome, proteopathy (protein conformation disease), cerebral β-amyloid angiopathy, retinal ganglion cell degeneration in glaucoma (retinal ganglion cell degeneration in glaucoma), synucleinopathy ( synucleinopathies), tauopathies, frontotemporal lobar degeneration (FTLD), dementia, Cadasil syndrome, hereditary cerebral hemorrhage with amyloidosis, Alexander disease, seipinopathies, familial amyloidotic neuropathy ), senile systemic amyloidosis (senile systemic amyloidosis), serpinopathies, AL (light chain) amyloidosis (primary systemic amyloidosis), AH (heavy chain) amyloidosis, AA (secondary ) Amyloidosis, Aortic Medial Amyloidosis, ApoAI Amyloidosis, ApoAII Amyloidosis, ApoAIV Amyloidosis, Finnish Type Familial Amyloidosis (FAF), Lysozyme Amyloidosis, Fibrinogen Amyloidosis denaturation, dialysis Powdery degeneration, inclusion body myositis/myopathy, cataract, retinitis pigmentosa with rhodopsin mutation, medullary thyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma, hereditary lattice corneal dystrophy, mossy skin Cutaneous lichen amyloidosis, Mallory bodies, corneal lactoferrin amyloidosis, alveolar proteinosis, odontogenic (Pindborg) tumor amyloid, cystic fibrosis, sickle Cellular disease or critical illness myopathy (CIM).

Detailed description of the invention

definition

The amino acid sequences of the invention contain the conventional one-letter and three-letter codes for naturally occurring amino acids, as well as the accepted three-letter codes for other amino acids, such as Aib (alpha-aminoisobutyric acid).

The term "natural exendin-4" refers to natural exendin-4 having the following sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS- _NH2 (SEQ ID NO: 4).

The present invention provides a peptide compound as defined above.

The peptidic compounds of the present invention comprise a linear backbone of aminocarboxylic acids linked by peptide, ie carboxamide, linkages. Preferably, the aminocarboxylic acid is an alpha-aminocarboxylic acid and more preferably an L-alpha-aminocarboxylic acid, unless otherwise specified. Preferably, the peptidic compound comprises a backbone sequence of 39 aminocarboxylic acids.

For the avoidance of doubt, in the definitions provided herein, it is generally intended that the sequence of the peptide moiety (Formula I) differs from native Exendin-4 at least 14 of those positions which are stated as allowing variation. The amino acids within the peptide module (Formula I) can be considered to be numbered consecutively from 1 to 39 in the conventional N-terminal to C-terminal direction. References to "positions" within the peptide module (I) should be interpreted accordingly, e.g. reference should be made to natural exendin-4 and other positions within the molecule, e.g., in exendin-4, His is at position 1 , Gly at 2nd, ..., Met at 14th, ... and Ser at 39th.

Amino acid residues with _-NH2 side chain groups, such as Lys, Orn, Dab or Dap, are functionalized because at least the H atom of the _-NH2 side chain group is replaced by -ZC(O) ^-R5 , where R ⁵ comprises a lipophilic moiety, such as an acyclic linear or branched (C ₈ -C ₃₀ ) saturated or unsaturated hydrocarbon group, which is unsubstituted or substituted, for example by halogen (F, Cl, Br, J), -OH and/or _CO2H substitution, and Z comprises linkers in all stereoisomeric forms, for example linkers comprising one or more, for example 1 to 5, preferably 1, 2 or 3 amino acid linker groups selected from the group consisting of: The groups γ-glutamic acid (gGlu) and AEEAc (amino-ethoxy-ethoxy-acetyl). Preferred groups R ⁵ comprise lipophilic moieties such as acyclic linear or branched (C ₁₂ -C ₂₀ ) saturated or unsaturated hydrocarbon groups such as pentadecyl, hexadecyl or heptadecyl, which are unsubstituted or substituted by _CO2H , more preferably pentadecyl or heptadecyl. In one embodiment, the amino acid linker group is selected from: gGlu, gGlu-gGlu, AEEAc-AEEAc-gGlu, and AEEAc-AEEAc-AEEAc. In another embodiment, the amino acid linker group is gGlu. In another embodiment, the amino acid linker group is gGlu-gGlu. In another embodiment, the amino acid linker group is AEEAc-AEEAc-gGlu. In another embodiment, the amino acid linker group is AEEAc-AEEAc-AEEAc.

In a further aspect, the invention provides compositions comprising a compound of the invention as described herein, or a salt or solvate thereof, in admixture with a carrier.

The present invention also provides the use of a compound of the invention for use as a medicament, in particular for the treatment of a condition as described in the specification.

The present invention further provides a method for the treatment of a condition as described herein comprising administering to a patient an effective amount of at least one compound of formula (I).

The present invention also provides compositions, wherein the composition is a pharmaceutically acceptable composition, and the carrier is a pharmaceutically acceptable carrier.

peptide synthesis

The skilled person knows many different methods to prepare the peptides described in the present invention. These methods include, but are not limited to, synthetic methods and recombinant gene expression. Thus, one way of preparing these peptides is synthesis in solution or on a solid support followed by isolation and purification. A different way of producing peptides is gene expression in host cells into which a DNA sequence encoding the peptide has been introduced. Alternatively, gene expression can be achieved without the use of cellular systems. The methods described above can also be combined in any way.

A preferred way of preparing the peptides of the invention is solid phase synthesis on a suitable resin. Solid phase peptide synthesis is a well established method (see for example: Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill., 1984; E. Atherton and R.C. Sheppard, Solid Phase Peptide Synthesis. A Practical Approach, Oxford-IRL Press, New York, 1989). Solid phase synthesis is initiated by attaching an N-terminally protected amino acid with its carboxyl terminus to an inert solid support bearing a cleavable linker. This solid support can be any polymer that allows the coupling of the initial amino acid, such as trityl resin, chlorotrityl resin, Wang resin (Wang resin) or Rink resin, wherein the carboxyl group (or for Rink The resin is a carboxamide) to the resin is acid sensitive (when using the Fmoc strategy). The polymeric support should be stable under the conditions used to deprotect the α-amino groups during peptide synthesis.

After the N-terminally protected first amino acid has been coupled to the solid support, the alpha-amino protecting group of this amino acid is removed. Then, the remaining protected Amino acids were coupled sequentially or with preformed dipeptides, tripeptides or tetrapeptides in the order indicated by the peptide sequence, where BOP, HBTU and HATU were used with a tertiary amine base. Alternatively, the released N-terminus can be functionalized with groups other than amino acids, such as carboxylic acids and the like.

Typically, the reactive side chain groups of amino acids are protected with suitable blocking groups. These protecting groups are removed after the desired peptide has been assembled. They are removed simultaneously with cleavage of the desired product from the resin under the same conditions. Protecting groups and protocols for introducing protecting groups can be found in Protective Groups in Organic Synthesis, 3rd Edition, Greene, T.W. and Wuts, P.G.M., Wiley & Sons (New York: 1999).

In some cases, it may be desirable to have side chain protecting groups that can be selectively removed while leaving other side chain protecting groups intact. In this case, the released functionality can be selectively functionalized. For example, lysine (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), the ivDde protecting group is unstable to very nucleophilic groups such as 4% hydrazine in DMF (dimethylformamide). Thus, if the N-terminal amino group and all side-chain functionality are protected with an acid-labile protecting group, the ivDde group can be selectively removed using 4% hydrazine in DMF, and the corresponding free group can be further modified, e.g., by acylation. amino group. Alternatively, lysine can be coupled to a protected amino acid, and the amino group of this amino acid can then be deprotected, resulting in another free amino group, which can be acylated or attached to other amino acids. Alternatively, prefunctionalized building blocks such as (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-( Hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid as coupling partner body, a side chain was introduced along with lysine during peptide synthesis (as described in Table 2). The use of this building block has the technical advantage that a selective deprotection step is not necessary and can avoid the selective attachment of side chain building blocks on very late synthetic intermediates.

Finally, the peptide is cleaved from the resin. This can be achieved by using the King mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude material can then be purified by chromatography, eg preparative RP-HPLC if necessary.

effectiveness

As used herein, the term "potency" or "in vitro potency" is a measure of the ability of a compound to activate receptors for GLP-1, glucagon or GIP in a cell-based assay. Numerically, it is expressed as an "EC50 value", which is the effective concentration of a compound that induces a half-maximal increase in response (eg formation of intracellular cAMP) in a dose-response experiment.

therapeutic use

Metabolic syndrome is a combination of medical conditions that when taken together increase the risk of developing type 2 diabetes, as well as atherosclerotic vascular disease, such as heart attack and stroke. Medical parameters defining the metabolic syndrome include diabetes mellitus, impaired glucose tolerance, elevated fasting glucose, insulin resistance, urinary albumin secretion, central obesity, hypertension, elevated triglycerides, elevated LDL cholesterol and reduced HDL cholesterol.

Obesity is a medical condition in which excess body fat has accumulated to such an extent that it can have adverse effects on health and life expectancy, and due to its increasing prevalence in adults and children it has become One of the leading preventable causes of death in the modern world. It increases the likelihood of a variety of other diseases, including heart disease, type 2 diabetes, obstructive sleep apnea, certain types of cancer, and osteoarthritis, and it is most often caused by excess food intake. Income, reduced energy expenditure, and a combination of genetic susceptibility.

Diabetes is a group of metabolic diseases in which an individual has high blood sugar levels either because the body does not produce enough insulin, or because cells do not respond to the insulin produced. The most common types of diabetes are: (1) type 1 diabetes, in which the body cannot produce insulin; (2) type 2 diabetes (T2DM), in which the body does not use insulin properly, in combination with insulin deficiency that increases over time, and (3) Gestational diabetes, in which a woman develops diabetes as a result of her pregnancy. All forms of diabetes increase the risk of long-term complications, which often develop over many years. Most of these long-term complications are based on damage to blood vessels and fall into two categories: "macrovascular" disease (which results from atherosclerosis of larger vessels) and "microvascular" disease (which results from damage to small blood vessels) ). Examples of macrovascular disease conditions are ischemic heart disease, heart failure, stroke and peripheral vascular disease. Examples of microvascular diseases are diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy.

The receptors for GLP-1 and GIP, as well as glucagon, are members of the family of 7 transmembrane-spanning, heterotrimeric G protein-coupled receptors. They are structurally related to each other and not only share considerable levels of sequence identity, but also share similar ligand recognition mechanisms and intracellular signaling pathways.

Similarly, the peptides GLP-1, GIP and glucagon share regions of high sequence identity/similarity. GLP-1 and glucagon are produced from a common precursor, preproglucagon, which is differentially processed in a tissue-specific manner to produce, for example, GLP-1 in enteroendocrine cells and alpha in pancreatic islets. Glucagon in cells. GIP is derived from the larger prohormone precursor proGIP and is synthesized and released from K cells located in the small intestine.

The peptide incretin hormones GLP-1 and GIP are secreted by enteroendocrine cells in response to food and account for as much as 70% of meal-stimulated insulin secretion. Evidence suggests that GLP-1 secretion is reduced in subjects with impaired glucose tolerance or type 2 diabetes, while responsiveness to GLP-1 is still preserved in these patients. Therefore, targeting the GLP-1 receptor with a suitable agonist provides an attractive approach for the treatment of metabolic disorders, including diabetes. The receptors of GLP-1 are widely distributed, mainly in pancreatic islets, brain, heart, kidney and gastrointestinal tract. In the pancreas, GLP-1 acts in a strictly glucose-dependent manner by increasing insulin secretion from β cells. This glucose dependence suggests that activation of the GLP-1 receptor is unlikely to cause hypoglycemia. Also, receptors for GIP are widely expressed in peripheral tissues including pancreatic islets, adipose tissue, stomach, small intestine, heart, bone, lung, kidney, testis, adrenal cortex, pituitary, endothelial cells, trachea, spleen, Thymus, thyroid and brain. Consistent with their biological function as incretin hormones, pancreatic beta cells express the highest levels of GIP receptors in humans. There is some clinical evidence that GIP receptor-mediated signaling can be impaired in T2DM patients, but it has been shown that GIP effects are reversible and can be restored with improvement of diabetic status. Although there are numerous reports of the effect of GIP on insulin secretion being also glucose dependent, there are also reports in the literature that high plasma levels of GIP can lead to more frequent hypoglycemic episodes (McLaughlin et al., J Clin Endocrinol 1983, 56, 648-652). In addition, plasma GIP levels were reported to be higher than normal in obese subjects, suggesting that GIP can induce obesity and insulin resistance (Creutzfeldt et al., Diabetologia. 1978, 14, 15-24). This is supported by reports that ablation of the GIP receptor can prevent those conditions: GIP receptor knockout mice fed a high-fat diet actually show suppression of body weight compared to wild-type mice (Miyawaki et al. , Nat Med. 2002, 8, 738-42), and chronic administration of the GIP receptor antagonist (Pro3) GIP also prevents obesity and insulin resistance in mice (Gault et al., Diabetologia. 2007, 50, 1752-62). It is therefore an object of the present invention to provide dual GLP-1/glucagon receptor agonists with reduced activity at the GIP receptor.

Glucagon is a 29 amino acid peptide hormone that is produced by pancreatic alpha cells and released into the bloodstream when circulating glucose is low. An important physiological role of glucagon is to stimulate glucose output in the liver, a process that provides insulin's major anti-regulatory mechanism in maintaining glucose homeostasis in vivo.

However, the glucagon receptor is also expressed in extrahepatic tissues such as kidney, heart, adipocytes, lymphoblastoids, brain, retina, adrenal gland, and gastrointestinal tract, suggesting a broader physiological role beyond glucose homeostasis. Thus, recent studies have reported therapeutically positive effects of glucagon on energy management, including stimulation of energy expenditure and thermogenesis, with concomitant reductions in food intake and weight loss. In conclusion, stimulation of the glucagon receptor may be useful in the treatment of obesity and metabolic syndrome.

Oxyntomodulin is a peptide hormone consisting of glucagon with 8 amino acids encompassing a C-terminal extension. Like GLP-1 and glucagon, it is preformed with preproglucagon and is cleaved and secreted in a tissue-specific manner by endocrine cells of the small intestine. Oxyntomodulin is known to stimulate both GLP-1 and glucagon receptors, and is thus a prototype of a dual agonist (see Pocai, Molecular Metabolism 2013; 3:241-51).

Since GLP-1 is known for its antidiabetic effect, both GLP-1 and glucagon are known for their food intake suppressing effect and glucagon is also a mediator of extra energy expenditure, it is conceivable that Combining two hormones in one molecule could lead to powerful drugs for treating metabolic syndrome and particularly its components diabetes and obesity.

Thus, the compounds of the present invention are useful in the treatment of glucose intolerance, insulin resistance, prediabetes, increased fasting glucose (hyperglycemia), type 2 diabetes, hypertension, dyslipidemia, arteriosclerosis, coronary heart disease, peripheral artery disease, Stroke or any combination of these individual disease components.

Additionally, they can be used to control appetite, food intake and calorie intake, increase energy expenditure, prevent weight gain, promote weight loss, reduce excess body weight and co-treat obesity, including morbid obesity.

The compounds of the present invention are agonists (e.g., "dual agonists") of the GLP-1 and glucagon receptors with reduced activity at the GIP receptor, and may provide therapeutic benefit to address metabolic syndrome for targeting According to the clinical needs of the symptoms, it is carried out by the simultaneous treatment of diabetes and obesity.

Other disease states and conditions that may be treated with the compounds of the invention are obesity-associated inflammation, obesity-associated gallbladder disease, and obesity-induced sleep apnea.

While all of these conditions may be directly or indirectly related to obesity, the effect of the compounds of the invention may be mediated in whole or in part via the effect on body weight or independent of it.

Furthermore, the disease to be treated is a neurodegenerative disease, such as Alzheimer's disease or Parkinson's disease, or other degenerative diseases as described above.

In one embodiment, the compounds are useful in the treatment or prevention of hyperglycemia, type 2 diabetes, obesity. In a preferred embodiment, the compounds are useful in the treatment of diabetes, especially type 2 diabetes.

Compounds of the invention have the ability to decrease intestinal transit, increase gastric content and/or decrease food intake in patients. These activities of the compounds of the invention can be assessed in animal models known to the skilled person and described herein in the Methods.

Compounds of the invention have the ability to lower blood glucose levels, and/or lower HbA1c levels in patients. These activities of the compounds of the invention can be assessed in animal models known to the skilled person and described herein in the Methods and also in Example 7.

The compounds of the present invention have the ability to reduce body weight in patients. These activities of the compounds of the invention can be assessed in animal models known to the skilled person and described herein in the Methods and also in Example 7.

The compounds of the present invention are useful in the treatment or prevention of hepatic steatosis, preferably nonalcoholic liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH).

Compared with GLP-1, glucagon and oxyntomodulin, exendin-4 has beneficial physicochemical properties, such as solubility and stability in solution and under physiological conditions (including against enzymes such as Enzymatic stability of the degradation of DPP4 or NEP), which results in a longer duration of action in vivo. Therefore, the pure GLP-1 receptor agonist exendin-4 could serve as a good starting scaffold to obtain exendin-4 analogs with dual GLP-1/glucagon receptor agonism.

However, exendin-4 has also been shown to be chemically unstable due to oxidation of methionine in position 14 and deamidation and isomerization of asparagine in position 28. Therefore, stability can be achieved by substituting methionine at position 14 and avoiding sequences known to be susceptible to degradation via aspartimide formation, especially Asp-Gly or Asn at positions 28 and 29. -Gly was further improved.

pharmaceutical composition

The term "pharmaceutical composition" refers to a mixture comprising ingredients which, when mixed, are compatible and can be administered. A pharmaceutical composition may contain one or more medicinal drugs. In addition, pharmaceutical compositions may contain carriers, buffers, acidifying agents, alkalizing agents, solvents, adjuvants, tonicity regulators, emollients, expanders, preservatives, physical and chemical stabilizers, such as surfactants , antioxidants and other components, whether these are considered active or inactive. Guidance for the skilled person in the preparation of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, (20th Ed.) A.R. Gennaro A.R. Ed., 2000, Lippencott Williams & Wilkins and R.C. Rowe et al. (Eds.), Handbook of Pharmaceutical Excipients, PhP , updated May 2013.

According to certain embodiments of the present invention there is provided a pharmaceutical composition comprising at least one compound of formula (I) or a physiologically acceptable salt or solvate of any of them.

The exendin-4 peptide derivatives or salts thereof of the present invention are administered together with pharmaceutically acceptable carriers, diluents, or excipients as part of a pharmaceutical composition. Also contemplated are solvates of compounds of general formula (I) together with pharmaceutically acceptable carriers, diluents or excipients as part of the pharmaceutical composition.

A "pharmaceutically acceptable carrier" is a carrier that is physiologically acceptable (eg, a physiologically acceptable pH) while retaining the therapeutic properties of the substance with which it is administered. Standard pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, (20th Ed.) A.R. Gennaro A.R. Ed., 2000, Lippencott Williams & Wilkins and R.C. Rowe et al. (Editors), Handbook of Pharmaceutical excipients, PhP, updated in May 2013. An exemplary pharmaceutically acceptable carrier is physiological saline solution.

In one embodiment, the carrier is selected from the group consisting of buffers (e.g. citrate/citric acid), acidifying agents (e.g. hydrochloric acid), alkalizing agents (e.g. sodium hydroxide), preservatives (e.g. phenol, meta cresol), co-solvents (eg polyethylene glycol 400), tonicity modifiers (eg mannitol), stabilizers (eg surfactants, antioxidants, amino acids).

The concentrations used are in the physiologically acceptable range.

Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal and transdermal) administration. Typically the compounds of the invention will be administered parenterally.

The term "pharmaceutically acceptable salt" means a salt of a compound of the present invention that is safe and effective for use in mammals. Pharmaceutically acceptable salts may include, but are not limited to, acid addition salts and basic salts. Examples of acid addition salts include chloride, sulfate, hydrogensulfate, (hydrogen)phosphate, acetate, citrate, tosylate or methanesulfonate, preferably acetate. Examples of basic salts include salts with inorganic cations, such as basic or alkaline earth metal salts, such as sodium, potassium, magnesium or calcium salts, and salts with organic cations, such as amine salts. Further examples of pharmaceutically acceptable salts are described in Remington: The Science and Practice of Pharmacy, (20th Edition) A.R. Gennaro A.R. Ed., 2000, Lippencott Williams & Wilkins or Handbook of Pharmaceutical Salts, Properties, Selection and Use, edited by P.H. Stahl, C.G. Wermuth, 2002, jointly published by Verlag Helvetica Chimica Acta, Zurich, Switzerland and Wiley-VCH, Weinheim, Germany.

The term "solvate" means a complex of a compound of the present invention or a salt thereof with solvent molecules, such as organic solvent molecules and/or water.

In the pharmaceutical composition, the exendin-4 derivatives may be in the form of monomers or oligomers.

The term "therapeutically effective amount" of a compound refers to a non-toxic but sufficient amount of the compound to provide the desired effect. The amount of compound of formula (I) necessary to achieve the desired biological effect depends on many factors such as the particular compound chosen, the intended use, the mode of administration and the clinical condition of the patient. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. For example, a "therapeutically effective amount" of a compound of formula (I) is about 0.01 to 50 mg/dose, preferably 0.02 to 1 mg/dose.

The pharmaceutical compositions of the invention are those suitable for parenteral (e.g. subcutaneous, intramuscular, intradermal or intravenous), rectal, topical or oral (e.g. sublingual) administration, although the most suitable administration The mode depends in each individual case on the nature and severity of the condition to be treated and on the nature of the compound of formula (I) used in each case. In one embodiment, the application is parenteral, eg subcutaneous.

In the case of parenteral applications, it may be advantageous for the respective formulations to contain at least one antimicrobial preservative in order to inhibit the growth of microorganisms and bacteria between administrations. Preferred preservatives are benzyl alcohol or phenolic compounds such as phenol or m-cresol. These ingredients have been described to reduce the aggregation of peptides and proteins, resulting in lower solubility and stability in formulations (see R.L.Bis et al., Int.J.Pharm.472, 356-361, 2014; T.J.Kamerzell, Adv.DrugDeliv . Rev., 63, 1118-1159, 2011).

Suitable pharmaceutical compositions may be in the form of discrete units in vials or ampoules, such as capsules, tablets and powders, each containing a defined amount of the compound; as a powder or granules; as a solution in an aqueous or non-aqueous liquid or suspensions; as oil-in-water or water-in-oil emulsions. It can be given in single or multi-dose injectable form, for example in the form of a pen. As already mentioned, the compositions may be prepared by any suitable method of pharmacy which comprises the step of bringing into contact the active ingredient and the carrier (which may consist of one or more further ingredients).

In certain embodiments, the pharmaceutical composition may be provided with an application device, for example, with a syringe, injection pen, or auto-injector. Such devices may be provided separate from or pre-filled with a pharmaceutical composition.

combination therapy

The compounds of the invention (dual agonists of GLP-1 and glucagon receptors) can be combined with other pharmacologically active compounds, such as all drugs mentioned in Rote Liste 2015, for example with Rote Liste 2015, Chapter 1 All weight-lowering agents or appetite suppressants, all lipid-lowering agents mentioned in Rote Liste 2015, chapter 58, all antihypertensives and nephroprotectants mentioned in Rote Liste 2015, or Rote Liste 2015, chapter 36 All diuretics mentioned in this chapter are combined extensively.

Active ingredient combinations can be used especially for a synergistic improvement of action. They can be applied by separate administration of the active ingredients to the patient or in the form of combination products in which several active ingredients are present in one pharmaceutical preparation. When the active ingredients are administered by separate administration of the active ingredients, this can be done simultaneously or sequentially.

Most of the active ingredients mentioned hereinafter are disclosed in the USP Dictionary of USAN and International Drug Names, US Pharmacopeia, Rockville 2011.

Other active substances suitable for such combinations include in particular those which, for example, intensify the therapeutic effect of one or more active substances and/or allow a reduction in the dose of one or more active substances for one of the indicated indications substance.

Accordingly, in certain embodiments, the present invention relates to a combination of a peptide compound of formula (I) and at least one additional therapeutically active agent.

Certain embodiments relate to pharmaceutical compositions comprising at least one compound of formula (I), or a physiologically acceptable salt or solvate of any of them, and at least one additional pharmaceutically active ingredient.

Therapeutic agents suitable for combination with compounds of formula (I) of the present invention include, for example, antidiabetic agents such as:

Insulin and insulin derivatives (insulin-like compounds), e.g. insulin glargine/ 270-330U/mL insulin glargine (EP 2387989A), 300U/mL insulin glargine ( EP2387989A), ^LAPS insulin-115, insulin glulisin (glulisin)/ Insulin detemir (detemir)/ Insulin Lispro (lispro)/ / Insulin degludec (degludec)/degludecPlus, insulin aspart (aspart), basal insulin and analogs (eg LY-2605541, LY2963016, NN1436), PEGylated insulin lispro, Linjeta, NN1045, insulin plus pramlintide acetate formulation (symlin), PE0139, rapid-acting and short-acting insulin (eg, Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhalable, transdermal, and sublingual Insulin (eg Afrezza, tregopil, TPM 02, Capsulin, Oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, Oshadi oral insulin). Also included are those insulin derivatives bound to albumin or another protein via a bifunctional linker.

GLP-1, GLP-1 analogues and GLP-1 receptor agonists, eg lixisenatide/AVE0010/ZP10/lyxumia, exenatide/exendin-4/Byetta/Bydureon /ITCA 650/AC-2993, liraglutide/Victoza, semaglutide, taspoglutide, syncria/albiglutide, dulaglutide, r Exendin-4 (recombinant exendin-4), CJC-1134-PC, PB-1023, TTP-054, Efpeglenatide/HM-11260C, CM-3, GLP-1Eligen, ORMD-0901, NN-9924, NN-9926, NN-9927, nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, TT-401, BHM-034. MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, Exenatide-XTEN (xtenized exenatide) and Glucagon-Xten (xtenized glucagon ). Optionally, GLP-1 and GLP-1 analogs can also be bound to the polymer.

DPP-4 (also known as DDP-IV or dipeptidyl peptidase IV) inhibitors such as: alogliptin/Nesina, Trajenta/linagliptin/BI-1356/Ondero/Trajenta /Tradjenta/Trayenta/Tradzenta, Saxagliptin/Onglyza, sitagliptin/Januvia/Xelevia/Tesave/Janumet/Velmetia, Galvus/vildagliptin, aragliptin ( anagliptin, gemigliptin, teneligliptin, melogliptin, trelagliptin, DA-1229, omarigliptin/MK-3102, KM-223, evogliptin, ARI- 2243, PBL-1427, pinoxacin.

SGLT2 (sodium glucose transporter 2) inhibitors such as: Invokana/canaglifozin, Forxiga/dapagliflozin, remoglifozin, sergliflozin, empagliflozin, ipagliflozin, topagliflozin Tofogliflozin, luseogliflozin, LX-4211, ertuglifozin/PF-04971729, RO-4998452, EGT-0001442, KGA-3235/DSP-3235, LIK066, SBM-TFC -039; Dual SGLT2/SGLT1 Inhibitor;

Biguanides (eg metformin, buformin, phenformin), thiazolidinediones (eg pioglitazone, rivoglitazone, rosiglitazone, troglitazone), Dual PPAR agonists (eg, aleglitazar, muraglitazar, tesaglitazar), sulfonylureas (eg, tolbutamide, glibenclamide, glimepiride /Amaryl, glipizide), meglitinides (such as nateglinide, repaglinide, mitiglinide), alpha-glucosidase inhibitors (such as Acarbose, miglitol, voglibose), amylin and amylin analogs (eg pramlintide, Symlin).

GPR119 (G protein-coupled receptor 119) agonists (eg GSK-263A, PSN-821, MBX-2982, APD-597, ZYG-19, DS-8500), GPR40 agonists (eg fasiglifam/TAK-875, TUG-424, P-1736, JTT-851, GW9508), GPR120 agonists, GPR142 agonists, systemic or low absorbable TGR5 (transmembrane G protein-coupled receptor 5) agonists.

Other suitable combination partners are: Cycloset (bromocriptine), 11-β-HSD inhibitors (11-β-hydroxysteroid dehydrogenase; for example LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD- 016, BI-135585), glucokinase activators (such as TTP-399, AMG-151, TAK-329, GKM-001), DGAT inhibitors (diacylglycerol acyltransferase; such as LCQ-908), protein tyrosine Amino acid phosphatase 1 inhibitors (eg, trodusquemine), glucose-6-phosphatase inhibitors, fructose-1,6-bisphosphatase inhibitors, glycogen phosphorylase inhibitors, phosphoenolpyruvate carboxykinase inhibitors , glycogen synthase kinase inhibitors, pyruvate dehydrogenase inhibitors, alpha2-antagonists, CCR-2 (C-C motif receptor 2) antagonists, SGLT-1 inhibitors (eg LX-2761), glucose transporters Protein-4 modulator, somatostatin receptor 3 agonist.

One or more lipid-lowering agents are also suitable as combination partners, such as for example: HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA)-reductase inhibitors (e.g. simvastatin ), atorvastatin), fibrates (eg, bezafibrate, fenofibrate), niacin and its derivatives (eg, niacin) , niacin receptor 1 agonist, PPAR (peroxisome proliferator-activated receptor)-(alpha, gamma or alpha/gamma) agonist or modulator (eg aleglitazar), PPAR- Delta agonists, ACAT (acyl-CoA cholesterol acyltransferase) inhibitors (such as avasimibe), cholesterol absorption inhibitors (such as ezetimibe), bile acid binding substances (such as cholera cholestyramine), ileal bile acid transport (IBAT) inhibitors, MTP (microsomal triglyceride transfer protein) inhibitors, or PCSK9 (proprotein convertase subtilisin/kexin type 9) modulators;

LDL (low density lipoprotein) receptor upregulators via liver selective thyroid hormone receptor beta agonists, HDL (high density lipoprotein) raising compounds such as: CETP inhibitors (eg torcetrapib, ancetrapid, dalcetrapid, evacetrapid, JTT-302, DRL-17822, TA-8995) or ABC1 modulators; lipid metabolism modulators; PLA2 inhibitors, ApoA-I (apolipoprotein A1) enhancers, Thyroid hormone receptor agonists, cholesterol synthesis inhibitors, omega-3 fatty acids and their derivatives.

Other suitable combination partners are one or more active substances for the treatment of obesity, such as for example: sibutramine, tesofensine, orlistat (tetrahydrogen clay) Polestatin (tetrahydrolipstatin), cannabinoid-1 receptor antagonist, MCH-1 (melanin concentrating hormone 1) receptor antagonist, MC4 (melanocortin 4) receptor agonist and partial agonist, NPY5 (neuropeptide Y5) or NPY2 antagonists (e.g. velneperit), NPY4 agonists, beta-3-agonists, leptin or leptin mimetics, 5HT2c receptor agonists (e.g. lorcaser lorcaserin), or bupropione/naltrexone (CONTRAVE), bupropion/zonisamide (EMPATIC), bupropion/phentermine or Pramlintide/metreleptin, or phentermine/topiramate (QNEXA) combination.

Other suitable combination partners are:

Other gastrointestinal peptides, such as peptide YY 3-36 (PYY3-36) or its analogs, pancreatic polypeptide (PP) or its analogs,

Glucagon receptor agonist or antagonist, GIP receptor agonist or antagonist, dual GLP-1/GIP agonist, dual GLP-1/glucagon agonist, Ghrelin antagonist or inverse agonist, Xenin and its analogs.

Other suitable combination partners are:

Lipase inhibitors, angiogenesis inhibitors, H3 antagonists, AgRP (Agouti-related protein) inhibitors, triple monoamine uptake inhibitors (norepinephrine and acetylcholine), MetAP2 (methionine aminopeptidase type 2) Inhibitors, nasal formulations of the calcium channel blocker diltiazem, antisense molecules generated against fibroblast growth factor receptor 4, inhibin targeting peptide-1.

Furthermore, in combination with drugs used to affect hypertension, chronic heart failure or atherosclerosis, such as, for example: angiotensin II receptor antagonists (eg telmisartan, candesartan, Valsartan, losartan, eprosartan, irbesartan, olmesartan, tasosartan, azilsartan ( azilsartan)), ACE (angiotensin-converting enzyme) inhibitors, ECE (endothelin-converting enzyme) inhibitors, diuretics, beta-blockers, calcium antagonists, centrally acting hypertensive drugs, alpha-2- Adrenergic receptor antagonists, neutral endopeptidase inhibitors, thrombin aggregation inhibitors and others or combinations thereof are suitable.

In a further aspect, the present invention relates to the use of a compound according to the invention or a physiologically acceptable salt thereof in combination with at least one active substance described above as combination partner for the preparation of a medicament suitable for the treatment of Or prevent a disease or condition that can be affected by binding to GLP-1 and the glucagon receptor and by modulating their activity. Preferably, this is a disease in the context of metabolic syndrome, in particular one of the diseases or conditions listed above, more in particular diabetes or obesity or complications thereof.

The use of the compounds according to the invention, or their physiologically acceptable salts, in combination with one or more active substances can take place simultaneously, separately or sequentially.

The combined use of a compound according to the invention, or a physiologically acceptable salt thereof, with another active substance can take place simultaneously or at staggered times, but in particular within short time intervals. If they are administered simultaneously, the patient is given both active substances together; if they are used at staggered times, the two active substances are given to the patient within a period of less than or equal to 12 hours, but in particular less than or equal to 6 hours.

Thus, in a further aspect, the invention relates to a medicament comprising a compound according to the invention or a physiologically acceptable salt of such a compound and at least one active substance described above as combination partner, optionally together with a One or more inert carriers and/or diluents.

The compound according to the invention, or its physiologically acceptable salt or solvate, and the other active substance to be combined therewith may be present together in one formulation, such as tablet or capsule, or separately in two identical Or in different formulations, for example as so-called kit-of-parts.

In a further aspect, the invention relates to a method for treating a condition as described herein comprising administering to a patient an effective amount of at least one compound of formula (I) and an effective amount of at least one other compound available for Compounds for the treatment of diabetes, dyslipidemia or hypertension. In certain embodiments, the condition is selected from hyperglycemia, type 2 diabetes, and obesity, and combinations thereof. In a particular embodiment, at least one other compound useful in the treatment of diabetes, dyslipidemia or hypertension is selected from the group consisting of the exemplary antidiabetic agents, lipid lowering agents, active substances for the treatment of obesity listed above , gastrointestinal peptides, and drugs used to affect high blood pressure, chronic heart failure, or atherosclerosis.

As already indicated previously, effective amounts of at least one compound of formula (I) and the further active ingredient may be administered to the patient simultaneously, or in an alternative sequentially.

Brief description of the drawings

Figure 1 : Body weight development during 4 weeks of subcutaneous treatment with SEQ ID NO: 6 and SEQ ID NO: 7, 50 μg/kg twice daily (bid) in female high fat fed C57BL/6 mice. Data are mean+SEM.

Figure 2: % change in relative body weight during 4 weeks of subcutaneous treatment with SEQ ID NO: 6 and SEQ ID NO: 7, 50 μg/kg twice daily in female high fat fed C57BL/6 mice. Data are mean+SEM.

Figure 3: Measurements by nuclear magnetic resonance (NMR) before and 2 days after 4 weeks of treatment with SEQ ID NO:6 and SEQ ID NO:7, 50 μg/kg bid in female high fat fed C57BL/6 mice Determination of total fat mass. Data are mean+SEM.

Figure 4: Short-term effects of s.c. administration of compounds SEQ ID NO:6 and SEQ ID NO:7 50 μg/kg on blood glucose in female high fat-fed C57BL/6 mice. Data are mean+SEM.

Figure 5a: Body weight development during 2 weeks in female high fat fed C57BL/6 mice treated subcutaneously with SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, 50 μg/kg twice a day. Data are mean+SEM.

Figure 5b: Body weight development during 2 weeks of subcutaneous treatment with SEQ ID NO: 11 and SEQ ID NO: 15, 50 μg/kg twice a day in female high fat fed C57BL/6 mice. Data are mean+SEM.

Figure 6a: Relative body weight % change during 2 weeks in female high fat fed C57BL/6 mice treated subcutaneously with SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, 50 μg/kg twice a day. Data are mean+SEM.

Figure 6b: Relative body weight % change during 2 weeks in female high fat fed C57BL/6 mice treated subcutaneously with SEQ ID NO: 11 and SEQ ID NO: 15, 50 μg/kg twice a day. Data are mean+SEM.

Figure 7a: Short-term effects of s.c. administration of compounds SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 50 μg/kg on blood glucose in female high fat-fed C57BL/6 mice. Data are mean+SEM.

Figure 7b: Short term effect on blood glucose of s.c. administration of compounds SEQ ID NO: 11 and SEQ ID NO: 15 50 μg/kg in female high fat fed C57BL/6 mice. Data are mean+SEM.

Figure 8a: Daily food consumption during 2 weeks in female high fat fed C57BL/6 mice treated subcutaneously with SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, 50 μg/kg twice a day.

Figure 8b: Daily food consumption during 2 weeks in female high fat fed C57BL/6 mice treated subcutaneously with SEQ ID NO: 11 and SEQ ID NO: 15, 50 μg/kg twice a day.

method

The following abbreviations are used:

AA amino acid

AEEAc (2-(2-Aminoethoxy)ethoxy)acetyl

Aib alpha-amino-isobutyric acid

cAMP cyclic adenosine monophosphate

Boc tert-butyloxycarbonyl

BOP (Benzotriazol-1-yloxy)tris(dimethylamino)phosphine hexafluorophosphate

BSA bovine serum albumin

tBu tert-butyl

dAla D-alanine

DCM dichloromethane

Dde 1-(4,4-Dimethyl-2,6-dioxocyclohexylene)-ethyl

ivDde 1-(4,4-Dimethyl-2,6-dioxocyclohexylene)-3-methyl-butyl

DIC N,N'-Diisopropylcarbodiimide

DIPEA N,N-Diisopropylethylamine

DMEM Dulbecco's Modified Eagle's Medium

DMF Dimethylformamide

DMS Dimethyl Sulfide

EDT Ethylenedithiol

FA formic acid

FBS fetal bovine serum

Fmoc Fluorenylmethoxycarbonyl

gGlu γ-glutamic acid (γE)

HATU O-(7-Azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (salt)

HBSS Hank's Balanced Salt Solution

HBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate (salt)

HEPES 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

HOBt 1-Hydroxybenzotriazole

HOSu N-Hydroxysuccinimide

HPLC high performance liquid chromatography

HTRF Homogeneous Time-Resolved Fluorescence

IBMX 3-isobutyl-1-methylxanthine

LC/MS Liquid Chromatography/Mass Spectrometry

Mmt monomethoxy-trityl

Palm Palmitoyl

PBS Phosphate Buffered Saline

PEG polyethylene glycol

PK pharmacokinetics

RP-HPLC Reversed Phase High Performance Liquid Chromatography

Stearoyl

TFA trifluoroacetic acid

Trt Trityl

UV ultraviolet light

General synthesis of peptide compounds

Material

Different Rink-amide resins (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucine were used at loadings ranging from 0.2-0.7 mmol/g Amidomethyl resin, Merck Biosciences; 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetamidomethyl resin, Agilent Technologies) for the synthesis of peptide amides.

Fmoc-protected natural amino acids were purchased from Protein Technologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, Iris Biotech, Bachem, Chem-Impex International or MATRIX Innovation. The following standard amino acids were used throughout the synthesis: Fmoc-L-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L -Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L -Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc- L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH.

Additionally, the following specific amino acids were purchased from the same suppliers as above: Fmoc-L-Lys(ivDde)-OH, Fmoc-L-Lys(Mmt)-OH, Fmoc-Aib-OH, Fmoc-D-Ser( tBu)-OH, Fmoc-D-Ala-OH, Boc-L-His(Boc)-OH (available as toluene solvate) and Boc-L-His(Trt)-OH.

In addition, applying the building block (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)- 5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid and Boc-L-His(Trt)- Aib-OH. Synthesize these two building blocks separately. Synthetic descriptions are given under the examples.

Solid phase peptide synthesis is performed, for example, on a Prelude peptide synthesizer (Protein Technologies Inc) or similar automated synthesizer using standard Fmoc chemistry and HBTU/DIPEA activation. DMF was used as solvent. Deprotection: 20% piperidine/DMF for 2x 2.5 minutes. Wash: 7x DMF. Coupling 2:5:10 DIPEA 2x in 200mM AA/500mM HBTU/2M DMF for 20 minutes. Wash: 5x DMF.

In the case of modification of the L-side chain, Fmoc-L-Lys(ivDde)-OH or Fmoc-L-Lys(Mmt)-OH is used in the corresponding position. After completion of the synthesis, the ivDde group was removed using 4% hydrazine hydrate in DMF according to a modified literature procedure (S.R. Chhabra et al., Tetrahedron Lett., 1998, 39, 1603). The Mmt group was removed by repeated treatment with 1% TFA in dichloromethane. Subsequent acylation is carried out by treating the resin with the N-hydroxysuccinimide ester of the desired acid or using a coupling reagent such as HBTU/DIPEA or HOBt/DIC.

All peptides that had been synthesized were cleaved from the resin with a King cleavage mix consisting of 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, 2.5% EDT. Crude peptides were then precipitated in diethyl ether or diisopropyl ether, centrifuged, and lyophilized. Peptides were analyzed by analytical HPLC and checked by ESI mass spectrometry. Crude peptides were purified by conventional preparative RP-HPLC purification procedures.

Alternatively, peptides were synthesized by a manual synthesis procedure: 0.3 g of dry Rink amide MBHA resin (0.66 mmol/g) was placed in a polyethylene container equipped with a polypropylene filter. The resin was swelled in DCM (15ml) for 1 hour and in DMF (15ml) for 1 hour. The Fmoc group on the resin was deprotected by treating it with 20% (v/v) piperidine/DMF solution twice for 5 and 15 minutes. Wash the resin with DMF/DCM/DMF (6:6:6 each). The removal of Fmoc from the solid support was confirmed using the Kaiser test (quantitative method). The C-terminal Fmoc-amino acid in dry DMF (5 equiv excess corresponding to resin loading) was added to the deprotected resin and the coupling of the next Fmoc-amino acid was initiated with 5 equiv excess of DIC and HOBT in DMF . The concentration of each reactant in the reaction mixture was about 0.4M. The mixture was rotated on a rotor for 2 hours at room temperature. The resin was filtered and washed with DMF/DCM/DMF (6:6:6 each). The Kaiser test on an aliquot of the peptide resin after complete coupling was negative (no color on the resin). After attachment of the first amino acid, unreacted amino groups (if any) in the resin were capped for 20 minutes using acetic anhydride/pyridine/DCM (1:8:8) to avoid any deletion of the sequence. After capping, the resin was washed with DCM/DMF/DCM/DMF (6/6/6/6 times each). The Fmoc group on the C-terminal amino acid-attached peptidyl resin was deprotected by treating it with 20% (v/v) piperidine/DMF solution twice for 5 and 15 minutes. Wash the resin with DMF/DCM/DMF (6:6:6 times each). The Kaiser test on an aliquot of the peptide resin after complete Fmoc deprotection was positive.

The remaining amino acids in the target sequence were sequentially coupled on Rink amide MBHA resin using the Fmoc AA/DIC/HOBt method using a 5 equivalent excess corresponding to the resin loading in DMF. The concentration of each reactant in the reaction mixture was about 0.4M. The mixture was rotated on a rotor for 2 hours at room temperature. The resin was filtered and washed with DMF/DCM/DMF (6:6:6 each). After each coupling step and Fmoc deprotection step, a Kaiser test was performed to confirm the completion of the reaction.

After completion of the linear sequence, the ε-amino group of lysine used as a branch point or modification point was deprotected by using 2.5% hydrazine hydrate in DMF for 15 minutes x 2, and washed with DMF/DCM/DMF ( 6:6:6 each). The γ-carboxyl terminus of glutamic acid was attached to the ε-amino group of Lys using Fmoc-Glu(OH)-OtBu in DMF by the DIC/HOBt method (5 equivalent excess relative to resin loading). The mixture was rotated in a rotor for 2 hours at room temperature. The resin was filtered and washed with DMF/DCM/DMF (6x30ml each). The Fmoc group on glutamic acid was deprotected by treating it with 20% (v/v) piperidine/DMF solution for 5 and 15 minutes (25 ml each). Wash the resin with DMF/DCM/DMF (6:6:6 times each). An aliquot of the peptide resin after complete Fmoc deprotection was positive for the Kaiser test on the resin.

If the side chain branch also contains 1 more γ-glutamic acid, a second Fmoc-Glu(OH)-OtBu is used to attach to Free amino group of γ-glutamic acid. The mixture was rotated in a rotor for 2 hours at room temperature. The resin was filtered and washed with DMF/DCM/DMF (6x30ml each). The Fmoc group on glutamic acid was deprotected by treating it with 20% (v/v) piperidine/DMF solution for 5 and 15 minutes (25 ml). Wash the resin with DMF/DCM/DMF (6:6:6 times each). An aliquot of the peptide resin after complete Fmoc deprotection was positive for the Kaiser test on the resin.

Attachment of palmitic and stearic acids to the side chain of glutamic acid:

Palmitic acid or stearic acid (5 equiv) dissolved in DMF was added to the free amino group of γ-glutamic acid and the coupling was initiated by adding DIC (5 equiv) and HOBt (5 equiv) in DMF . Wash the resin with DMF/DCM/DMF (6:6:6 times each).

Finally the peptide is cleaved from the resin:

The peptidyl resin synthesized by manual synthesis was washed with DCM (6x10ml), MeOH (6x10ml) and ether (6x10ml) and dried overnight in a vacuum desiccator. Peptide cleavage from the solid support was achieved by treating the peptide-resin with a reagent mixture (80.0% TFA/5% thioanisole/5% phenol/2.5% EDT, 2.5% DMS and 5% DCM) for 3 hours at room temperature. The cleavage mixture was collected by filtration and washed with TFA (2ml) and DCM (2x 5ml). Excess TFA and DCM were concentrated to a small volume under nitrogen, and a small amount of DCM (5-10 ml) was added to the residue and evaporated under nitrogen. Repeat the process 3-4 times to remove most of the volatile impurities. The residue was cooled to 0°C, and anhydrous ether was added to precipitate the peptide. The precipitated peptide was centrifuged and the supernatant ether was removed and fresh ether was added to the peptide and centrifuged again. The crude peptide was purified by preparative HPLC and lyophilized. Peptide identities were confirmed by LCMS.

Alternatively, prefunctionalized building blocks with side chains already attached to lysines (e.g. (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S) )-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluorene-9- A different route for the introduction of lysine side chains was used as coupling partner. 0,67 mmol of peptide resin carrying amino groups was washed with 20 ml of dimethylformamide. Together with 310 mg of hydroxybenzotriazole hydrate and 0,32 ml of diisopropylcarbodiimide, 2,93 g of (2S)-6-[[(4S)-5-tert-butoxy-4-[[( 4S)-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluorene-9 -Hydroxymethoxycarbonylamino)hexanoic acid was dissolved in 20ml of dimethylformamide. After stirring for 5 minutes, the solution was added to the resin. The resin was stirred for 20 hours and then washed three times with 20 ml each of dimethylformamide. Small resin samples were taken and subjected to Kaiser and Chloranil tests (Kaiser, Colescott, Bossinger, Cook, Anal. Biochem. 1970, 34, 595-598; Chloranil-Test: Vojkovsky, Peptide Research 1995, 8, 236-237). This procedure avoids the need for selective deprotection steps and selective attachment of side chain building blocks on very late synthetic intermediates.

Analytical HPLC/UPLC

Method A: Detection at 210-225nm

Column: Waters ACQUITY CSH ^TM C18 1.7μm (150x 2.1mm) at 50℃

Solvent: H ₂ O+0.5%TFA:ACN+0.35%TFA (flow rate 0.5ml/min)

Gradient: 80:20 (0 minutes) to 80:20 (3 minutes) to 25:75 (23 minutes) to 2:98 (23.5 minutes) to 2:98 (30.5 minutes) to 80:20 (31 minutes) to 80:20 (37 minutes)

Optionally with mass analyzer: LCT Premier, electrospray positive ion mode

Method B: Detection at 210-225nm

Column: Aries prep XBC 18 (4.6x250mm x 3.6μm), Temp: 25°C

Solvent: H ₂ O+0.1% TFA (buffer A): ACN+0.1% TFA (flow rate 1ml/min) (buffer B)

Gradient: The column was equilibrated with 2% buffer B and eluted by a gradient of 2% to 70% buffer B during 15 minutes.

Method C: Detection at 214nm

Column: Waters ACQUITY CSH ^TM C18 1.7μm (150x 2.1mm) at 50°C

Solvent: H ₂ O+0.5%TFA:ACN+0.35%TFA (flow rate 0.5ml/min)

Gradient: 80:20 (0 minutes) to 80:20 (3 minutes) to 25:75 (23 minutes) to 5:95 (23.5 minutes) to 5:95 (25.5 minutes) to 80:20 (26 minutes) to 80:20 (30 minutes)

General Preparative HPLC Purification Protocol

exist Crude peptides were purified on a Purifier System, a Jasco semiprep HPLC System or an Agilent 1100 HPLC system. Depending on the amount of crude peptide to be purified, different sizes of preparative RP-C18-HPLC columns and different flow rates were used. Acetonitrile + 0.1% TFA (B) and water + 0.1% TFA (A) were used as eluents. Fractions containing product are pooled and lyophilized to obtain purified product, usually the TFA salt.

Solubility and Stability Evaluation of Exendin-4 Derivatives

Purity of peptide batches was determined via UPLC/MS prior to solubility and stability measurements.

For solubility testing, the target concentration was 10 mg pure compound/ml. Therefore, based on the previously determined % purity, solutions from the solid samples were prepared in two different buffer systems with a concentration of 10 mg/mL compound:

a) Acetate buffer pH4.5, 100mM sodium acetate trihydrate, 2.7mg/ml m-cresol

b) Phosphate buffer pH7.4, 100mM sodium hydrogen phosphate, 2.7mg/ml m-cresol

UPLC-UV was performed after 2 hours of gentle stirring from the supernatant obtained after centrifugation at 3000 RCF (relative centrifugal acceleration) for 20 minutes.

Solubility was determined by comparing the UV peak area of 2 μL injections of buffered samples diluted 1:10 with a standard curve. Standards were dilutions (various volumes ranging from 0.2-2 μl by injection) of peptides at a concentration of 1.2 mg/mL (based on % purity) in DMSO stock solutions. The sample also served as the starting point (t0) for stability testing.

For the chemical and physical stability tests, aliquots of the supernatant obtained for solubility were stored at 40°C for 7 days. After the time course, samples were centrifuged at 3000 RCF for 20 minutes. Then, 2 μl of the 1:10 diluted supernatant was analyzed by UPLC-UV.

Chemical stability is graded via the relative loss of purity calculated by the following equation:

[(purity at starting point)-(purity after 7 days)]/(purity at starting point)]*100%

Purity is calculated as:

[(peak area peptide)/(total peak area)]*100%

In addition, the remaining amount of the peptide was determined by comparing the peak area of the target peptide after 7 days with the peak area at the starting point. This leads to the parameter "% remaining peptide", following the equation:

% remaining peptide = [(peak area peptide t7)/(peak area peptide t0)]*100%.

Dynamic Light Scattering (DLS)

Dynamic Light Scattering (DLS) measures the light scattered from particles based on Brownian motion (the interaction of particles with solvent molecules). A liquid sample is illuminated with a focused laser beam, and time-dependent scattered light intensity fluctuations are recorded and correlated. The first order result from a DLS experiment is the intensity distribution of the particle size. The intensity distribution is naturally weighted according to the scattering intensity of each particle fraction or family. For biological materials or polymers, the particle scattering intensity is proportional to the square of the molecular weight. The mean hydrodynamic diameter of the particles present in the sample is directly related to the rate of change of the scattered light intensity and is calculated using the Stokes-Einstein relationship. The polydispersity index is a measure of the breadth of the size distribution and is calculated by standard methods documented in ISO13321:1996 and ISO22412:2008.

Method A: DLS measurements were performed on a W130i device (Avid Nano Ltd, High Wycombe, UK) and using low volume disposable cuvettes (UVette, Eppendorf AG, Hamburg, Germany). Data were processed with i-Size 3.0 provided by Avid Nano. The hydrodynamic diameter was determined by the non-negatively constrained least squares (NNLS) method using the DynaLS algorithm. Measured at a 90° angle.

Method B: DLS measurements were performed on a Nanosizer ZS (Malvern Instruments, Malvern, UK) and using disposable UV cuvettes (Brand macro, 2,5 mL and Brand semi-micro 1,5 mL, Brand GmbH+Co KG, Wertheim, Germany) . Data were processed with Malvern Zetasizer software version 7.10 or 7.01. The hydrodynamic diameter was determined by non-negative constrained least squares using the DynaLS algorithm. Measurements were performed in NIBS (Non-Invasive Back-Scatter) mode at an angle of 173°.

Method C: DLS measurements were performed on a DynaPro plate reader II (Wyatt Technology, Santa Barbara, CA, US) and using low volume, non-treated, black polystyrene 384 assay plates with clean bottoms (Corning, NY, US) . Data were processed with Dynamics V 7.1.9.3 provided by Wyatt Technology. The hydrodynamic diameter was determined by non-negative constrained least squares using the DynaLS algorithm. Measurements were made with an 830nm laser light source at an angle of 158°.

In Vitro Cellular Assays for GLP-1, Glucagon and GIP Receptor Potency

Agonism of the receptor by compounds was determined by a functional assay measuring the cAMP response of HEK-293 cell lines stably expressing human GLP-1, GIP or glucagon receptors.

The cAMP content of cells was determined based on HTRF (Homogeneous Time-Resolved Fluorescence) using a kit from Cisbio Corp. (Catalog No. 62AM4PEJ). For preparation, cells were split into T175 culture flasks and grown overnight in (DMEM/10% FBS) until nearly confluent. Then, the medium was removed, and the cells were washed with calcium- and magnesium-deficient PBS, followed by protease treatment with Accutase (Sigma-Aldrich Cat# A6964). Detached cells were washed and resuspended in assay buffer (1x HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX) and cell density was determined. They were then diluted to 400000 cells/ml and 25 μl aliquots were dispensed into wells of a 96-well plate. For the measurement, 25 μl of test compound in assay buffer was added to the wells, followed by incubation at room temperature for 30 minutes. After addition of HTRF reagent diluted in lysis buffer (kit component), the plate was incubated for 1 hour, followed by measurement of the fluorescence ratio at 665/616 nm. In vitro potency of agonists is quantified by determining the concentration at which 50% of the activation elicits a maximal response (EC50).

Bioanalytical Screening Method for Quantification of Exendin-4 Derivatives in Mice and Pigs

1 mg/kg was administered subcutaneously (s.c.) to mice. Mice were sacrificed and blood samples were collected 0.25, 0.5, 1, 2, 4, 8, 16 and 24 hours after application. Plasma samples were analyzed after protein precipitation via liquid chromatography mass spectrometry (LC/MS). PK parameters and half-lives were calculated using WinonLin Version 5.2.1 (non-compartmental model).

to female Minipigs were administered subcutaneously (sc) at 0.05 mg/kg, 0.075 mg/kg or 0.1 mg/kg. Blood samples were collected 0.25, 0.5, 1, 2, 4, 8, 24, 32, 48, 56 and 72 hours after application. Plasma samples were analyzed after protein precipitation via liquid chromatography mass spectrometry (LC/MS). PK parameters and half-lives were calculated using WinonLin Version 5.2.1 (non-compartmental model).

Gastric emptying and intestinal passage in mice

Female NMRI mice weighing 20-30 g were used. Mice were acclimated to housing conditions for at least one week.

Mice were fasted overnight, but water was kept available. On study day, mice were weighed and housed individually, and allowed to have access to 500 mg chow for 30 minutes, but water was removed. At the end of the 30 minute feeding period, the remaining feed was removed and weighed. After 60 minutes, a colored non-caloric bolus was instilled via gastric gavage. Test compound/reference compound or its vehicle in a control group is administered subcutaneously to achieve Cmax when a colored bolus is administered. After a further 30 minutes, the animals were sacrificed, and the stomach and small intestine were prepared. The full stomach was weighed, emptied, carefully cleaned, dried and reweighed. The calculated gastric contents indicate the degree of gastric emptying. The small intestine was straightened without force and measured in length. Then, measure the distance from the stomach start of the intestine to the tip of the furthest traveling intestinal content bolus. Intestinal passage is given as a percentage of the latter distance and the total length of the small intestine.

Comparable data can be obtained for both female and male mice.

Statistical analysis was performed by 1-way ANOVA with Everstat 6.0, followed by Dunnetts or Newman-Keuls as post hoc tests, respectively. Differences were considered statistically significant at the p<0.05 level. As a post hoc test, Dunnet's test was applied for comparison with vehicle-only controls. The Newman-Keul test was applied for all pairwise comparisons (ie relative to vehicle and reference groups).

Automated assessment of feed intake in mice

Female NMRI mice weighing 20-30 g were used. Mice were acclimated to housing conditions for at least one week and housed individually in the evaluation facility for at least one day while baseline data were recorded. On study days, test products were administered subcutaneously close to the light-off period (12 hours light-off), and the assessment of feed consumption began directly thereafter. Assessments consisted of continuous monitoring (every 30 minutes) over 22 hours. It is possible to repeat this procedure over several days. Limiting the assessment to 22 hours was for practical purposes to allow re-weighing of animals, refilling of food and water, and administration of drugs between procedures. Results can be evaluated as accumulated data over 22 hours or differentiated into 30 minute intervals.

Comparable data can be obtained for both female and male mice.

Statistical analysis was performed by two-way ANOVA on repeated measures with Dunnett's post hoc test using Everstat 6.0. Differences were considered statistically significant at the p<0.05 level.

Short- and long-term effects of subcutaneous treatment on blood glucose and body weight in female diet-induced obese (DIO) C57BL/6 mice.

C57BL/6 Harlan mice were housed in a specific pathogen-free barrier facility on a 12-hour light/dark cycle with free access to water and a standard or high-fat diet. After 16 hours pre-ingestion of the high fat diet, mice were sacrificed into treatment groups (n=8) such that each group had a similar mean body weight. An age-matched group exposed to a standard chow arbitrarily was included as a standard control group. Mice were injected subcutaneously (s.c.) with vehicle solution and weighed for 3 days to acclimate them to the protocol before starting treatment.

1) Short-term effects on blood glucose in fed female DIO mice: immediately after the first administration (subcutaneously) of vehicle (phosphate buffered saline) or exendin-4 derivative (dissolved in phosphate buffered saline The initial blood sample was collected before the infusion. The administration volume was 5 mL/kg. Animals were exposed to water and their corresponding diet during the experiment. Blood glucose levels were measured at t = 0 hours, t = 1 hour, t = 2 hours, t = 3 hours, t = 4 hours, t = 6 hours and t = 24 hours (Method: Accu-Check blood glucose meter ). Blood sampling was performed by tail excision without anesthesia.

2) Long-term effects on body weight in female DIO mice: Mice were treated subcutaneously with vehicle or exendin-4 derivatives twice daily for 4 weeks at the beginning and end of the light phase, respectively, in the morning and in the evening . Body weight was recorded daily. Total fat mass was measured by nuclear magnetic resonance (NMR) 2 days before starting treatment and on day 26.

Statistical analysis was performed with Everstat 6.0 by repeated measures two-way ANOVA with Dunnetts post-hoc analysis (glucose profile) and one-way ANOVA followed by Dunnetts post-hoc test (body weight, body fat). Differences from vehicle-treated DIO control mice were considered statistically significant at the p<0.05 level.

Effects of 4-week Treatment on Glucose, HbA1c, and Oral Glucose Tolerance in Female Diabetic dbdb Mice

Female diabetic dbdb mice with mean non-fasting glucose values 10-30 mmol/l and body weight 35-50 g were used. Mice were individually labeled and acclimatized to housing conditions for at least one week.

7 days before the start of the study, the baseline values of non-fasting glucose and HbA1c were measured, and 5 days before the start of the study, the mice were assigned to groups and cages according to their HbA1c values (5 mice per cage, 10 mice per group) to ensure Uniform distribution of lower and upper values across groups (stratification).

Mice were treated by twice daily subcutaneous administration in the morning and afternoon for 4 weeks. Blood samples from the tail tip were obtained for HbA1c on study day 21 and oral glucose tolerance was assessed in week 4.

Oral glucose tolerance testing was done in the morning without prior additional compound administration to primarily assess long-term treatment and effects and lesser effects of short-term compound administration. Mice were fasted for 4 hours before oral glucose administration (2 g/kg, t=0 min). Blood samples were drawn prior to glucose administration and at 15, 30, 60, 90, 120 and 180 minutes. Feed was returned after the last blood sampling. Results are expressed as change from baseline, glucose in mmol/l and HbA1c in %.

Statistical analysis was performed with SAS-based Everstat Version 6.0 by 1-way ANOVA followed by Dunnett's post hoc test relative to vehicle control. Differences were considered statistically significant at the p<0.05 level.

Glucose reduction in nonfasting female diabetic dbdb mice

Female diabetic dbdb mice with mean non-fasting glucose values 20-30 mmol/l and body weight 35-50 g were used. Mice were individually labeled and acclimatized to housing conditions for at least one week.

3-5 days before the start of the study, mice were assigned to groups and cages according to their non-fasting glucose values (4 mice per cage, 8 per group) to ensure lower and higher values between groups (stratification) of even distribution. On study day, mice were weighed and dosed (t=0). Feed was removed immediately prior to compound administration, but water remained available, and the first blood sample (baseline) was drawn from the tail nick. Additional blood samples were drawn at the tail incision at 30, 60, 90, 120, 240, 360 and 480 minutes.

Statistical analysis was performed with SAS-based Everstat Version 6.0 by 2-way ANOVA on repeated measures followed by Dunnett's post hoc test against vehicle control. Differences were considered statistically significant at the p<0.05 level.

Example

The invention is further illustrated by the following examples.

Example 1:

Synthesis of SEQ ID NO:6

In Novabiochem Rink-amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh , loaded at 0.23 mmol/g to perform the solid-phase synthesis described in Methods. The Fmoc synthesis strategy was applied in the presence of HBTU/DIPEA activation. Fmoc-Lys(ivDde)-OH in position 14 and Boc-His(Trt)-OH in position 1 were used in the solid phase synthesis protocol. The ivDde group was cleaved from the peptide on the resin according to the literature (S.R. Chhabra et al., Tetrahedron Lett. 1998, 39, 1603-1606). In the following, Palm-gGlu-gGlu-OSu was coupled to the released amino group using DIPEA as a base. Peptides were cleaved from resins with King mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified by preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μM) using an acetonitrile/water gradient (both buffers with 0,1% TFA). The purified peptides were analyzed by LCMS (Method A).

Deconvolution of the mass signal found under the peak with retention time 11.30 minutes revealed a peptide mass of 4782.6 consistent with the expected value of 4782.6.

Example 2:

Synthesis of SEQ ID NO:7

In Novabiochem Rink-amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl value), 100-200 mesh , loaded at 0.23 mmol/g to perform the solid-phase synthesis described in Methods. The Fmoc synthesis strategy was applied in the presence of HBTU/DIPEA activation. Fmoc-Lys(ivDde)-OH in position 14 and Boc-His(Trt)-OH in position 1 were used in the solid phase synthesis protocol. The ivDde group was cleaved from the peptide on the resin according to the literature (S.R. Chhabra et al., Tetrahedron Lett. 1998, 39, 1603-1606). In the following, Palm-gGlu-gGlu-OSu was coupled to the released amino group using DIPEA as a base. Peptides were cleaved from resins with King mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 1990, 36, 255-266). The crude product was purified by preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μM) using an acetonitrile/water gradient (both buffers with 0,1% TFA). The purified peptides were analyzed by LCMS (Method A).

Deconvolution of the mass signal found under the peak with retention time 10.77 minutes revealed a peptide mass of 4768.6 consistent with the expected value of 4768.6.

In a similar manner, the peptides listed in Table 3 were synthesized and characterized.

Table 3: List of synthetic peptides and comparison of calculated and found molecular weights

Examples 3a and 3b: Modification of the natural synthesis of SEQ ID NO:6 and SEQ ID NO.7.

Example 3a: Synthesis of building block (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino )-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid

Suspend 20g of O1-tert-butyl O5-(2,5-dioxypyrrolidin-1-yl)(2S)-2-[[(4S)-5-tert-butoxy- 4-(Hexadecanoylamino)-5-oxo-pentanoyl]amino]glutarate (obtained from WuXi AppTec). In parallel, 12 g of Fmoc-Lys-OH were suspended in 100 ml of ethyl acetate, and 13 ml of ethyldiisopropylamine were added. The two fractions were combined followed by additional ethyl acetate (50ml) and stirred at room temperature. After stirring for 3 hours, an additional 5 ml of ethyldiisopropylamine was added. The suspension was stirred for 20 hours. The suspension was filtered and extracted twice each with 200 ml of 10% citric acid solution followed by one extraction with 200 ml of brine. The organic layer was dried over sodium sulfate. Sodium sulfate was filtered and the solution was evaporated to dryness.

The remaining solid was taken up with 560ml methanol and 140ml water was added slowly. After stirring for 4 hours, the precipitate was filtered and washed once with 100 ml methanol/water (4:1). The product was dried under vacuum yielding 21 g of (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoyl Amino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid. Calculated MW = 976.5 g/mol, MW found [M+H] ⁺ = 977.5 g/mol.

Synthesis of Building Block Boc-L-His(Trt)-Aib-OH

2.5 g Boc-L-His(Trt)-OH was suspended in 25 ml ethyl acetate, and 1.55 g H-Aib-OBzl xHCl and 2,3 g HBTU were added, followed by 2,6 ml N-ethylmorpholine (N- ethylmorpholin). The suspension was stirred for 4.5 hours. Then, the mixture was extracted three times with 20 ml of 8% sodium bicarbonate solution, followed by one wash with water. The phases were separated and the organic layer was dried over sodium sulfate. After removal of the desiccant, the solution was evaporated to dryness and 4.5 g of a yellow oil was obtained consisting of Boc-L-His(Trt)-Aib-OBzl with >90% purity.

Dissolve 4.3 g Boc-L-His(Trt)-Aib-OBzl in 30 ml THF and 3 ml ethyl acetate. 0.43 g of hydrogenation catalyst Pd/C (5%) was added and the compound was hydrogenated at 30°C for 2 hours under atmospheric hydrogen pressure using a balloon. The catalyst was filtered off and washed with 30 ml ethyl acetate. The solution was stirred for several hours until precipitation occurred. The precipitate was filtered off and dried under vacuum to obtain 2.45 g of Boc-L-His(Trt)-Aib-OH.

These building blocks can be used to synthesize SEQ ID NO:6, SEQ ID No:7 or other corresponding peptides as described in the Methods.

Using the building block (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5- Oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid and with the building block Boc-L-His(Trt) -Aib-OH Synthesis of SEQ ID NO:6

On Agilent Rink-amide resin 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetamidomethyl resin at a loading of 0,27 mmol/g as described in the method solid-phase synthesis. The Fmoc synthesis strategy was applied in the presence of HBTU/DIPEA activation. (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)- 5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid was activated in three Double excess coupling, and the coupling time is controlled until the ninhydrin test shows negative results (Kaiser, Colescott, Bossinger, Cook, Analytical Biochemistry 34, 1970, 595ff). For positions 1 and 2, the building block Boc-L-His(Trt)-Aib-OH was also applied in a three-fold excess with HBTU/DIPEA activation, and the coupling time was controlled until the ninhydrin test showed negative results . Peptides were cleaved from the resin with King mixture (King, Fields, Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). A crude product was obtained (average MW calculated = 4785, 6 g/mol; average MW found = 4785, 7 g/mol). Crude peptides were purified as described under Methods.

Example 3b: Using building block (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino )-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid and with the building block Boc-L- His(Trt)-Aib-OH Synthesis SEQ ID NO:7

On Agilent Rink-amide resin 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetamidomethyl resin at a loading of 0,27 mmol/g as described in the method solid-phase synthesis. The Fmoc synthesis strategy was applied in the presence of HBTU/DIPEA activation. Position 14(2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5- Oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid in three-fold excess with HBTU/DIPEA activation Coupling was performed and the coupling time was controlled until the ninhydrin test showed negative results (Kaiser, Colescott, Bossinger, Cook, Analytical Biochemistry 1970, 34, 595-598). For positions 1 and 2, the building block Boc-L-His(Trt)-Aib-OH was also applied in a three-fold excess with HBTU/DIPEA activation, and the coupling time was controlled until the ninhydrin test showed negative results . Peptides were cleaved from the resin with King mixture (King, Fields, Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was obtained (average calculated MW=4771,6 g/mol; average found 4771.3 g/mol). Crude peptides were purified as described under Methods.

Example 4: Stability and Solubility

Peptide compounds were assessed for solubility and stability as described in Methods. The results are given in Table 4.

Table 4: Stability and Solubility

Example 5: In vitro data on GLP-1, glucagon and GIP receptors

By exposing cells expressing the human glucagon receptor (hglucagon R), human GIP receptor (hGIP-R), or human GLP-1 receptor (hGLP-1R) to increasing concentrations of the listed Compounds, and cAMP formed were measured to determine the potency of peptide compounds at GLP-1, glucagon and GIP receptors as described in Methods.

The results are shown in Table 5:

Table 5: EC50 values of exendin-4 derivatives on GLP-1, glucagon and GIP receptors (in pM)

SEQ ID NO EC50hGLP-1R EC50h Glucagon-R EC50hGIP-R 6 3.2 66.2 4170.0 7 2.1 24.0 1964.3 8 3.0 57.2 2310.0 9 4.2 22.2 1080.0 10 4.7 29.1 1100.0 11 6.9 58.9 2210.0 12 3.3 53.2 2880.0 13 6.4 22.3 1810.0 14 10.2 19.4 2870.0 15 5.5 33.5 3580.0 16 6.0 208.0 6740.0 17 9.0 149.0 8540.0 18 1.2 3.2 193.0 19 1.8 5.9 148.0 20 6.8 5.8 86.1

Embodiment 6: comparative test

The compounds carrying positions 27 and 34 have been tested relative to corresponding compounds having amino acid residues (Lys27, Ser32, Gly34, Ala35, Ser39) and otherwise identical amino acid sequences of native exendin-4 in these positions. A selection of inventive exendin-4 derivatives of Aib amino acid, Pro at position 32 and Lys at position 35 and 39 in . Table 6 gives the corresponding EC50 values (in pM) for the reference pairs of compounds and at the GLP-1, glucagon and GIP receptors. As shown, the inventive exendin-4 derivatives exhibit reduced activity at the GIP receptor compared to the corresponding derivatives which carry the same amino acid, maintaining its GLP-1R activity.

Table 6: Exendin-4 Derivatives Carrying Aib Amino Acids in Positions 27 and 34, Pro in Position 32 and Lys in Positions 35 and 39 and Natural Exendin Contained at These Positions Comparison of the amino acid residues of -4 (Lys27, Ser32, Gly34, Ala35, Ser39) and exendin-4 derivatives of the otherwise identical amino acid sequence. EC50 values at GLP-1, glucagon and GIP receptors are indicated in pM.

Example 7: SEQ ID NO:6 after subcutaneous treatment in female diet-induced obese (DIO) C57BL/6 mice and the short-term and long-term effects of SEQ ID NO:7 on blood glucose and body weight.

1) Glucose profile

Following blood sampling to determine baseline blood glucose levels, diet-induced obese female C57BL/6 mice were subcutaneously administered 50 μg/kg SEQ ID NO:6, 50 μg/kg SEQ ID NO:7 or phosphate buffered saline (standard or Vehicle control on high fat diet). At pre-defined time points, further blood samples are taken to measure blood glucose and generate a 24 hour blood glucose profile. SEQ ID NO:6 and SEQ ID NO:7 show a significant reduction in blood glucose compared to DIO control mice for time points t=1, 2, 3, 4, 6 and 24 hours after compound administration (p<0.0001 , 1-W-ANOVA-RM, post-hoc Dunnett's test; Figure 4, mean ± SEM).

2) weight

Female obese C57BL/6 mice were treated subcutaneously with 50 μg/kg SEQ ID NO: 6, 50 μg/kg SEQ ID NO: 7 or vehicle twice daily for 4 weeks. Body weight was recorded daily and body fat content was determined before the start of treatment and after 4 weeks of treatment. Treatment with 50 μg/kg of SEQ ID NO:6 showed a statistically significant reduction in daily body weight when compared to vehicle DIO control mice, beginning on day 7 and continuing to the end of the study (by end of study, p< 0.0001). Treatment with 50 μg/kg of SEQ ID NO:7 significantly reduced body weight when compared to vehicle DIO control mice, beginning on day 5 and continuing to the end of the study (p<0.0001 by end of study, Table 7, Fig. 1 and 2). These changes resulted from a reduction in body fat, as shown by absolute changes in body fat content (Table 7, Figure 3).

Table 7: Weight change in DIO mice over the 4 week treatment period (mean ± SEM)

example (dosage) Overall weight change (g) Body fat change (g) control standard diet +2.17±1.93 +0.25±0.34 control high fat diet +7.44±1.07 +2.39±0.43 SEQ ID NO:6 (50μg/kg twice a day) -11.71±1.45 -4.44±0.47 SEQ ID NO:7 (50μg/kg twice a day) -8.45±3.2 -3.83±1.38

Example 8: SEQ ID NO following subcutaneous treatment in female diet-induced obese (DIO) C57BL/6 mice: 8. Short-term effects of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:15 on blood glucose and body weight and long-term effects.

1) Glucose profile

Following blood sampling to determine baseline blood glucose levels, diet-induced obese female C57BL/6 mice were subcutaneously administered 50 μg/kg SEQ ID NO:8, 50 μg/kg SEQ ID NO:9, 50 μg/kg SEQ ID NO: 10 (Figure 7a), 50 μg/kg SEQ ID NO: 11, 50 μg/kg SEQ ID NO: 15 (Figure 7b) or phosphate buffered saline (vehicle control on standard or high fat diet). At pre-defined time points, further blood samples are taken to measure blood glucose and generate a 24 hour blood glucose profile.

All compounds showed a significant reduction in blood glucose compared to DIO control mice for time points t=1, 2, 3, 4, 6 and 24 hours after compound administration (t=0 hours, p<0.0001, 1-W - ANOVA-RM, post hoc Dunnett's test; Fig. 7a and b, means ± SEM).

1) Body weight and food consumption

With 50 μg/kg SEQ ID NO:8, 50 μg/kg SEQ ID NO:9 and 50 μg/kg SEQ ID NO:10 or vehicle (Figure 5a and 6a) and 50 μg/kg SEQ ID NO:11 and 50 μg/kg, respectively Female obese C57BL/6 mice were treated subcutaneously with SEQ ID NO: 15 or vehicle (Figures 5b and 6b) twice daily for 2 weeks.

Body weight and food consumption were recorded daily.

All compounds showed a statistically significant reduction in daily body weight when compared to vehicle DIO control mice, beginning on day 6 and continuing to the end of the study (p<0.0001 by the end of the study, Table 8, Figures 5a and b and 6a and b). These changes arose from an initial decrease in food consumption and were maintained despite resumption of food consumption over extended periods of time (Figures 8a and 8b).

Table 8: Weight change in DIO mice over the 2-week treatment period (strains ± SEM)

example (dosage) Overall weight change (g) control standard diet +1.0±0.3 control high fat diet +4.3±0.6 SEQ ID NO:8 (50μg/kg twice a day) -1.66±0.5 SEQ ID NO:9 (50μg/kg twice a day) -2.5±0.2 SEQ ID NO:10 (50μg/kg twice a day) -2.3±0.6 SEQ ID NO:11 (50μg/kg twice a day) -2.1±0.6 SEQ ID NO:15 (50μg/kg twice a day) -2.6±0.5

Table 9: Sequence

sequence listing

<110> SANOFI (Sanofi)

<120> New exendin-4 derivatives as selective peptide dual GLP-1/glucagon receptor agonists

<130> DE2015/082 WO PCT / 60324P WO

<150> EP 15 306 141.1

<151> 2015-07-10

<160> 20

<170> PatentIn version 3.5

<210> 1

<211> 30

<212> PRT

<213> Human (Homo sapiens)

<220>

<221>MOD_RES

<222> (30)..(30)

<223> Amidated C-terminus

<400> 1

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210> 2

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> Lycilla

<220>

<221>MOD_RES

<222> (20)..(20)

<223> Lys derivatized at N6 with (S)-4-carboxy-4-hexadecanoylamino-butyl

<400> 2

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

20 25 30

<210> 3

<211> 29

<212> PRT

<213> Human (Homo sapiens)

<400> 3

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 4

<211> 39

<212> PRT

<213> Gila monster (Heloderma suspectum)

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 4

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 5

<211> 42

<212> PRT

<213> Human (Homo sapiens)

<400> 5

Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys

20 25 30

Lys Asn Asp Trp Lys His Asn Ile Thr Gln

35 40

<210> 6

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> At N6 use (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butylamino)-

Butyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (29)..(29)

<223> D-Ala

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 6

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ala Xaa Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 7

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> At N6 use (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butylamino)-

Butyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 7

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ala Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 8

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> At N6 use (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butylamino)-

Butyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 8

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Lys Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 9

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> Lys derivatized at N6 with (S)-4-carboxy-4-octadecanoylamino-butyl

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 9

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ala Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 10

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> Lys derivatized at N6 with (S)-4-carboxy-4-octadecanoylamino-butyl

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 10

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Lys Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 11

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> Lys derivatized at N6 with (S)-4-carboxy-4-hexadecanoylamino-butyl

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 11

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ala Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 12

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> Use (2-{2-[2-(2-{2-[(4S)-4-carboxy-4-hexadecanoylamino-butylamino]-

Ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 12

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ala Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 13

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> Use (2-{2-[2-(2-{2-[(4S)-4-carboxy-4-octadecanoylamino-butylamino]-

Ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 13

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ala Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 14

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> Use [2-(2-{2-[2-(2-{2-[2-(2-octadecanoylamino-ethoxy)-ethoxy)-ethoxy]-acetylamino at N6

}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 14

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ala Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 15

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> At N6 use (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butylamino)-

Butyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 15

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ser Gly Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 16

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> At N6 use (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butylamino)-

Butyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (29)..(29)

<223> D-Ala

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 16

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Lys Xaa Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 17

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> At N6 use (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butylamino)-

Butyl derivatized Lys

<220>

<221>MOD_RES

<222> (27)..(27)

<223> Aib

<220>

<221>MOD_RES

<222> (29)..(29)

<223> D-Ala

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Aib

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 17

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Xaa Ser Xaa Gly Pro Pro

20 25 30

Ser Xaa Lys Pro Pro Pro Lys

35

<210> 18

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> At N6 use (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butylamino)-

Butyl derivatized Lys

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 18

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Lys Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 19

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> At N6 use (S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butylamino)-

Butyl derivatized Lys

<220>

<221>MOD_RES

<222> (29)..(29)

<223> D-Ala

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 19

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Lys Ala Xaa Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 20

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> exendin-4 analogs

<220>

<221>MOD_RES

<222> (2)..(2)

<223> Aib

<220>

<221>MOD_RES

<222> (14)..(14)

<223> Lys derivatized at N6 with (S)-4-carboxy-4-octadecanoylamino-butyl

<220>

<221>MOD_RES

<222> (39)..(39)

<223> Amidated C-terminus

<400> 20

His Xaa Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Lys Asp Glu

1 5 10 15

Gln Arg Ala Lys Leu Phe Ile Glu Trp Leu Lys Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

Claims (42)

1. one kind has the peptide compounds or its salt or solvate of formula (I)：

H₂N-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Asp-Glu-Gln- Arg-Ala-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Aib-X28-X29-Gly-Pro-Pro-Ser-Aib-Lys-Pro- Pro-Pro-Lys-R¹ (I)

Wherein

X14 represents there is functionalization-NH₂The amino acid residue of side-chain radical, it is selected from the group：Lys, Orn, Dab or Dap, wherein - the NH₂Side-chain radical passes through-Z-C (O)-R⁵Functionalization, wherein

Z represents the connector of all stereoisomeric forms in any ratio, and

R⁵It is to include up to 50 carbon atoms and the heteroatomic module selected from N and O,

X28 represents the amino acid residue selected from Ala, Lys and Ser,

X29 represents the amino acid residue selected from D-Ala and Gly,

R¹It is NH₂Or OH.

2. the compound or its salt or solvate of claim 1,

Wherein R¹It is NH₂。

3. the compound of any one of claim 1 to 2,

Wherein described peptide compounds are on glucagon receptor with least 0.09% phase compared with natural glucagon To activity.

4. the compound of any one of claims 1 to 3,

Wherein described peptide compounds shown on GLP-1 acceptors compared with GLP-1 (7-36)-acid amides at least 0.1% it is opposite Activity.

5. the compound of any one of claims 1 to 4, wherein

X14 represents Lys, wherein-the NH₂Side-chain radical group-Z-C (O) R⁵Functionalization, wherein

Z represents group selected from the group below：GGlu, gGlu-gGlu, AEEAc-AEEAc-gGlu and AEEAc-AEEAc-AEEAc, and And

R⁵Represent group selected from the group below：Pentadecyl or heptadecyl.

6. the compound or its salt or solvate of any one of claim 1 to 5, wherein

X14 represents Lys, wherein-the NH₂Side-chain radical passes through following functionalization：(S) -4- carboxyls -4- hexadecanoyl groups amino-fourth Acyl group -, (S) -4- carboxyl -4- octadecanoyls amino-butyryl -, (S) -4- carboxyls -4- ((S) -4- carboxyl -4- hexadecanoyl group ammonia Base-bytyry amino)-bytyry -, (2- { 2- [2- (2- { 2- [(4S) -4- carboxyl -4- hexadecanoyl group amino-butyryl ammonia Base]-ethyoxyl }-ethyoxyl)-acetyl-amino]-ethyoxyl }-ethyoxyl)-acetyl group-, (2- 2- [2- (2- 2- [(4S)- 4- carboxyl -4- octadecanoyl amino-butyryls amino]-ethyoxyl }-ethyoxyl)-acetyl-amino]-ethyoxyl }-ethoxy Base)-acetyl group-, [2- (2- 2- [2- (2- { 2- [2- (2- octadecanoyls Amion-ethoxy)-ethyoxyl]-acetyl-amino }- Ethyoxyl)-ethyoxyl]-acetyl-amino }-ethyoxyl)-ethyoxyl]-acetyl group-,

X28 represents Ala,

X29 represents the amino acid residue selected from D-Ala and Gly,

R¹Represent NH₂。

7. the compound or its salt or solvate of any one of claim 1 to 6, wherein

X14 represents Lys, wherein-the NH₂Side-chain radical passes through (S) -4- carboxyls -4- ((S) -4- carboxyl -4- hexadecanoyl group ammonia Base-bytyry amino)-bytyry-functionalization,

X28 represents Ser,

X29 represents the amino acid residue selected from D-Ala and Gly,

R¹Represent NH₂。

8. the compound or its salt or solvate of any one of claim 1 to 7, wherein

X14 represents Lys, wherein-the NH₂Side-chain radical passes through (S) -4- carboxyls -4- ((S) -4- carboxyl -4- hexadecanoyl group ammonia Base-bytyry amino)-bytyry -, (S) -4- carboxyl -4- octadecanoyls amino-butyryl-functionalization,

X28 represents Lys,

X29 represents the amino acid residue selected from D-Ala and Gly,

R¹Represent NH₂。

9. the compound or its salt or solvate of any one of claim 1 to 8, wherein

X14 represents Lys, wherein-the NH₂Side-chain radical passes through (S) -4- carboxyls -4- ((S) -4- carboxyl -4- hexadecanoyl group ammonia Base-bytyry amino)-bytyry-functionalization,

X28 represents the amino acid residue selected from Ala, Lys and Ser,

X29 represents D-Ala,

R¹Represent NH₂。

10. the compound or its salt or solvate of any one of claim 1 to 9, wherein

X14 represents Lys, wherein-the NH₂Side-chain radical passes through following functionalization：(S) -4- carboxyls -4- hexadecanoyl groups amino-fourth Acyl group -, (S) -4- carboxyl -4- octadecanoyls amino-butyryl -, (S) -4- carboxyls -4- ((S) -4- carboxyl -4- hexadecanoyl group ammonia Base-bytyry amino)-bytyry -, (2- { 2- [2- (2- { 2- [(4S) -4- carboxyl -4- hexadecanoyl group amino-butyryl ammonia Base]-ethyoxyl }-ethyoxyl)-acetyl-amino]-ethyoxyl }-ethyoxyl)-acetyl group-, (2- 2- [2- (2- 2- [(4S)- 4- carboxyl -4- octadecanoyl amino-butyryls amino]-ethyoxyl }-ethyoxyl)-acetyl-amino]-ethyoxyl }-ethoxy Base)-acetyl group-, [2- (2- 2- [2- (2- { 2- [2- (2- octadecanoyls Amion-ethoxy)-ethyoxyl]-acetyl-amino }- Ethyoxyl)-ethyoxyl]-acetyl-amino }-ethyoxyl)-ethyoxyl]-acetyl group-,

X28 represents the amino acid residue selected from Ala, Lys and Ser,

X29 represents Gly,

R¹Represent NH₂。

11. the compound or its salt or solvate of any one of claims 1 to 10, wherein

X14 represents Lys, wherein-the NH₂Side-chain radical passes through (S) -4- carboxyls -4- ((S) -4- carboxyl -4- hexadecanoyl group ammonia Base-bytyry amino)-bytyry-functionalization,

X28 represents Ala,

X29 represents the amino acid residue selected from Gly and D-Ala,

R¹Represent NH₂。

12. the compound or its salt or solvate of any one of claim 1 to 11, wherein

X14 represents Lys, wherein-the NH₂Side-chain radical passes through (S) -4- carboxyls -4- ((S) -4- carboxyl -4- hexadecanoyl group ammonia Base-bytyry amino)-bytyry-functionalization,

X28 represents the amino acid residue selected from Ala, Ser and Lys,

X29 represents the amino acid residue selected from Gly and D-Ala,

R¹Represent NH₂。

13. the compound of any one of claim 1 to 12 and its salt or solvate, it is selected from SEQ ID NO:6-17's Compound.

14. the compound or its salt or solvate of claim 1, wherein the compound is with SEQ ID NO.:6 represent.

15. the compound or its salt or solvate of claim 1, wherein the compound is with SEQ ID NO.:7 represent.

16. the compound or its salt or solvate of claim 1, wherein the compound is with SEQ ID NO.:8 represent.

17. the compound or its salt or solvate of claim 1, wherein the compound is with SEQ ID NO.:9 represent.

18. the compound or its salt or solvate of claim 1, wherein the compound is with SEQ ID NO.:10 represent.

19. the compound or its salt or solvate of claim 1, wherein the compound is with SEQ ID NO.:11 represent.

20. the compound or its salt or solvate of claim 1, wherein the compound is with SEQ ID NO.:15 represent.

21. the compound of any one of claim 1 to 20, it is used for medicine, particularly human medical.

22. the compound of any one of claim 1 to 20, it is used as medicine.

23. the compound of the purposes for any one of claim 21 to 22, it can as activating agent and at least one medicine Receive carrier together in pharmaceutical composition to exist.

24. the compound of the purposes for any one of claim 21 to 23, it exists with least one other therapeutically active agent Together.

25. the compound of the purposes for claim 24, wherein the other therapeutically active agent of at least one is selected from the group：

Insulin and insulin derivates；

GLP-1, GLP-1 analog and GLP-1 receptor stimulating agents；

DPP-4 inhibitor；

SGLT2 inhibitor；

Dual SGLT2/SGLT1 inhibitor；

Biguanides, thiazolinedione, dual PPAR agonist, sulfonylureas, meglitinides, Alpha-glucosidase inhibitor, shallow lake Powder not lysin (amylin) and amylin analog；

GPR119 activators, GPR40 activators, GPR120 activators, GPR142 activators, systematicness or low absorbability TGR5 Activator；

Bromocriptine (bromocriptine mesylate), 11- β-HSD inhibitor, activators of glucokinase, DGAT inhibitor, 1 inhibitor of protein tyrosine phosphatase, G-6-Pase inhibitor, fructose-1,6-diphosphonic acid enzyme inhibitor, glycogen Phosphorglase inhibitor, phosphoenolpyruvate carboxykinase inhibitor, glycogen synthase kinase enzyme inhibitor, pyruvate dehydrogenase kinases (pyruvate dehydrokinase) inhibitor, α 2- antagonists, CCR-2 antagonists, SGLT-1 inhibitor, glucose transport Conditioning agent, 3 activator of the somatostatin receptor of protein-4；

Lipid lowering agent；

For treating the active material of obesity；

Gastrointestinal peptide；

Lipase inhibitor, angiogenesis inhibitor, H3 antagonists, AgRP inhibitor, triple monoamine uptake inhibitors, MetAP2 Inhibitor, the nose preparaton of calcium channel blocker diltiazem, the antisense point for fibroblast growth factor receptor 4 generation Son, inhibin (prohibitin) targeting peptides -1；With

For influencing hypertension, chronic heart failure or the medicine of atherosclerosis.

26. the compound of the purposes for any one of claim 21 to 25, it is used to treat glucose intolerance, insulin Resistance, prediabetes, increased fasting glucose, hyperglycemia, diabetes B, hypertension, dyslipidemia, artery sclerosis, Coronary heart disease, peripheral arterial disease, any combinations of apoplexy or these other disease components.

27. the compound of the purposes for any one of claim 21 to 25, it is used to controlling appetite, ingest and calorie is taken the photograph (calory intake) is taken, increases energy expenditure, prevents weight increase, promotes weight saving, reducing excessive weight, and it is fat, Treatment together including morbid obesity.

28. the compound of the purposes for any one of claim 21 to 25, it is used to treat or prevent fatty degeneration of liver (hepatosteatosis)。

29. the compound of the purposes for any one of claim 21 to 25, it is used to treat or prevent hyperglycemia, 2 types Diabetes, obesity.

30. the compound of the purposes for any one of claim 21 to 25, it is used to treat diabetes and obesity at the same time.

31. the compound of the purposes for any one of claim 21 to 25, it is used to treat diabetes.

32. the compound of the purposes for any one of claim 21 to 25, it, which is used to reducing intestines, passes through (intestinal Passage), increase gastric content and/or reduce the food intake of patient.

33. the compound of the purposes for any one of claim 21 to 25, its be used to reducing blood glucose levels and/or The HbA1c for reducing patient is horizontal.

34. the compound of the purposes for any one of claim 21 to 25, it is used for the weight for reducing patient.

35. a kind of pharmaceutical composition, it includes any at least one compound of any one of claim 1 to 20 or they The physiology acceptable salt or solvate of kind, said composition are used as medicine.

36. a kind of pharmaceutical composition, it includes any at least one compound of any one of claim 1 to 20 or they The physiology acceptable salt or solvate of kind.

37. a kind of pharmaceutical composition, it includes appoint at least one compound of such as any one of claim 1 to 20 or they A kind of physiology acceptable salt or solvate and at least one other medicating active ingredients.

38. a kind of method of hyperglycemia for being used to treat in patient, diabetes B or obesity, the described method includes to described Patient applies the compound and effectively at least one formula (I) being claimed in any one of a effective amount of claim 1 to 20 At least one other compound that can be used for treatment diabetes, obesity, dyslipidemia or hypertension of amount.

39. the method for claim 38, wherein the patient is administered simultaneously a effective amount of at least one formula (I) compound and Other active ingredient.

40. the method being claimed in claim 38, wherein sequential to the patient apply a effective amount of at least one formula (I) Compound and other active ingredient.

41. the compound being claimed in claim 1 to 20, it is synthesized via synthesis in solid state, wherein using building block (2S) -6- [[(4S) -5- t-butoxies -4- [[(4S) -5- t-butoxies -4- (hexadecanoyl group amino) -5- oxos-valeryl Base] amino] -5- oxo-pentanoyls] amino] -2- (9H- fluorenes -9- methoxycarbonylamino) caproic acid.

Building block 42. (2S) -6- [[(4S) -5- t-butoxies -4- [[(4S) -5- t-butoxies -4- (hexadecanoyl group amino) - 5- oxo-pentanoyls] amino] -5- oxo-pentanoyls] amino] -2- (9H- fluorenes -9- bases-methyloxycarbonylamino) caproic acid.